26,27-homologated-20-epi-2-alkyl-19-nor-vitamin D compounds

ABSTRACT

This invention provides a novel class of vitamin D related compounds, namely, the 2-alkyl-19-nor-vitamin D derivatives, as well as a general method for their chemical synthesis. The compounds have the formula:  
                 
 
     where Y 1  and Y 2 , which may be the same or different, are each selected from the group consisting of hydrogen and a hydroxy-protecting group, R 6  is selected from the group consisting of alkyl, hydroxyalkyl and fluoroalkyl, and where the group R represents any of the typical side chains known for vitamin D type compounds. These 2-substituted compounds, especially the 2α-methyl and the 2α-methyl-20S derivatives, are characterized by relatively high intestinal calcium transport activity and relatively high bone calcium mobilization activity resulting in novel therapeutic agents for the treatment of diseases where bone formation is desired, particularly low bone turnover osteoporosis. These compounds also exhibit pronounced activity in arresting the proliferation of undifferentiated cells and inducing their differentiation to the monocyte thus evidencing use as anti-cancer agents and for the treatment of diseases such as psoriasis.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of application Ser.No. 09/454,013 filed Dec. 3, 1999, which is a divisional of applicationSer. No. 09/135,463 filed Aug. 17, 1998, which is a Continuation-In-Partof application Ser. No. 08/819,694 filed Mar. 17, 1997, now U.S. Pat.No. 5,945,410.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with United States government supportawarded by the following agencies:

[0003] NIH DK 14881-26S1

[0004] The United States has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0005] This patent invention relates to vitamin D compounds, and moreparticularly to vitamin D derivatives substituted at the carbon 2position.

[0006] The natural hormone, 1α,25-dihydroxyvitamin D₃ and its analog inergosterol series, i.e. 1α,25-dihydroxyvitamin D₂ are known to be highlypotent regulators of calcium homeostasis in animals and humans, and morerecently their activity in cellular differentiation has beenestablished, Ostrem et al., Proc. Natl. Acad. Sci. USA, 84, 2610 (1987).Many structural analogs of these metabolites have been prepared andtested, including 1α-hydroxyvitamin D₃, 1α-hydroxyvitamin D₂, variousside chain homologated vitamins and fluorinated analogs. Some of thesecompounds exhibit an interesting separation of activities in celldifferentiation and calcium regulation. This difference in activity maybe useful in the treatment of a variety of diseases such as renalosteodystrophy, vitamin D-resistant rickets, osteoporosis, psoriasis,and certain malignancies.

[0007] Recently, a new class of vitamin D analogs has been discovered,i.e. the so called 19-nor-vitamin D compounds, which are characterizedby the replacement of the A-ring exocyclic methylene group (carbon 19),typical of the vitamin D system, by two hydrogen atoms. Biologicaltesting of such 19-nor-analogs (e.g., 1α,25-dihydroxy-19-nor-vitamin D₃)revealed a selective activity profile with high potency in inducingcellular differentiation, and very low calcium mobilizing activity.Thus, these compounds are potentially useful as therapeutic agents forthe treatment of malignancies, or the treatment of various skindisorders. Two different methods of synthesis of such 19-nor-vitamin Danalogs have been described (Perlman et al., Tetrahedron Lett. 31, 1823(1990); Perlman et al., Tetrahedron Lett. 32, 7663 (1991), and DeLuca etal., U.S. Pat. No. 5,086,191).

[0008] In U.S. Pat. No. 4,666,634, 2β-hydroxy and alkoxy (e.g., ED-71)analogs of 1α,25-dihydroxyvitamin D₃ have been described and examined byChugai group as potential drugs for osteoporosis and as antitumoragents. See also Okano et al., Biochem. Biophys. Res. Commun. 163 1444(1989). Other 2-substituted (with hydroxyalkyl, e.g., ED-120, andfluoroalkyl groups) A-ring analogs of 1α,25-dihydroxyvitamin D₃ havealso been prepared and tested (Myamoto et al., Chem. Pharm. Bull. 41,1111 (1993), Nishii et al., Osteoporosis Int. Suppl. 1, 190 (1993);Posner et al., J. Org. Chem. 59 7855 (1994), and J. Org. Chem. 60g, 4617(1995)).

[0009] Recently, 2-substituted analogs of 1α,25-dihydroxy-19-norvitaminD₃ have also been synthesized, i.e. compounds substituted at 2-positionwith hydroxy or alkoxy groups (DeLuca et al., U.S. Pat. No. 5,536,713),which exhibit interesting and selective activity profiles. All thesestudies indicate that binding sites in vitamin D receptors canaccommodate different substituents at C-2 in the synthesized vitamin Danalogs.

[0010] In a continuing effort to explore the 19-nor class ofpharmacologically important vitamin D compounds, their analogs which arecharacterized by the presence of an alkyl (particularly methyl)substituent at the carbon 2 (C-2), i.e. 2-alkyl-19-nor-vitamin Dcompounds, and particularly 2-methyl-19-nor-vitamin D compounds, havenow been synthesized and tested. Such vitamin D analogs seemedinteresting targets because the relatively small alkyl (particularlymethyl) group at C-2 should not interfere with binding to the vitamin Dreceptor. On the other hand it is obvious that a change of conformationof the cyclohexanediol ring, A can be expected for these new analogs.

BRIEFS SUMMARY OF THE INVENTION

[0011] A class of 1α-hydroxylated vitamin D compounds not knownheretofore are the 19-nor-vitamin D analogs having an alkyl(particularly methyl) group at the 2-position, i.e.2-alkyl-19-nor-vitamin D compounds, particularly 2-methyl-19-nor-vitaminD compounds.

[0012] Structurally these novel analogs are characterized by the generalformula I shown below:

[0013] where Y₁ and Y₂, which may be the same or different, are eachselected from the group consisting of hydrogen and a hydroxy-protectinggroup, R₆ is selected from the group consisting of alkyl, hydroxyalkyland fluoroalkyl, and where the group R represents any of the typicalside chains known for vitamin D type compounds.

[0014] More specifically R can represent a saturated or unsaturatedhydrocarbon radical of 1 to 35 carbons, that may be straight-chain,branched or cyclic and that may contain one or more additionalsubstituents, such as hydroxy- or protected-hydroxy groups, fluoro,carbonyl, ester, epoxy, amino or other heteroatomic groups. Preferredside chains of this type are represented by the structure below

[0015] where the stereochemical center (corresponding to C-20 in steroidnumbering) may have the R or S configuration, (i.e. either the naturalconfiguration about carbon 20 or the 20-epi configuration), and where Zis selected from Y, —OY, —CH₂OY, —C≡CY, —CH═CHY, and —CH₂CH₂CH═CR³R⁴,where the double bond may have the cis or trans geometry, and where Y isselected from hydrogen, methyl, —COR⁵ and a radical of the structure:

[0016] where m and n, independently, represent the integers from 0 to 5,where R¹ is selected from hydrogen, deuterium, hydroxy, protectedhydroxy, fluoro, trifluoromethyl, and C₁₋₅-alkyl, which may be straightchain or branched and, optionally, bear a hydroxy or protected-hydroxysubstituent, and where each of R², R³, and R⁴, independently, isselected from deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyland C₁₋₅ alkyl, which may be straight-chain or branched, and optionally,bear a hydroxy or protected-hydroxy substituent, and where R¹ and R²,taken together, represent an oxo group, or an “allylidene group, =CR²R³,or the group —(CH₂)_(p)—, where p is an integer from 2 to 5, and whereR³ and R⁴, taken together, represent an oxo group, or the group—(CH₂)_(q)—, where q is an integer from 2 to 5, and where R⁵ representshydrogen, hydroxy, protected hydroxy, C₁₋₅ alkyl or —OR⁷ where R⁷represents C₁₋₅ alkyl, and wherein any of the CH-groups at positions 20,22, or 23 in the side chain may be replaced by a nitrogen atom, or whereany of the groups —CH(CH₃)—, —CH(R³)—, or —CH(R²)— at positions 20, 22,and 23, respectively, may be replaced by an oxygen or sulfur atom.

[0017] The wavy lines to the substituents at C-2 and at C-20 indicatethat the carbon 2 and carbon 20 may have either the R or Sconfiguration.

[0018] Specific important examples of side chains with natural20R-configuration are the structures represented by formulas (a), b),(c), (d) and (e) below. i.e. the side chain as it occurs in25-hydroxyvitamin D₃ (a); vitamin D₃ (b); 25-hydroxyvitamin D₂ (c);vitamin D₂ (d); and the C-24 epimer of 25-hydroxyvitamin D₂ (e):

[0019] Specific important examples of side chains with the unnatural 20S(also referred to as the 20-epi) configuration are the structurespresented by formulas (f) and (g) below:

[0020] The above novel compounds exhibit a desired, and highlyadvantageous, pattern of biological activity. These compounds arecharacterized by relatively high intestinal calcium transport activity,as compared to that of 1α,25-dihydroxyvitamin D₃, while also exhibitingrelatively high activity, as compared to 1α,25-dihydroxyvitamin D₃, intheir ability to mobilize calcium from bone. Hence, these compounds arehighly specific in their calcemic activity. Their preferential activityon mobilizing calcium from bone and either high or normal intestinalcalcium transport activity allows the in vivo administration of thesecompounds for the treatment of metabolic bone diseases where bone lossis a major concern. Because of their preferential calcemic activity onbone, these compounds would be preferred therapeutic agents for thetreatment of diseases where bone formation is desired, such asosteoporosis, especially low bone turnover osteoporosis, steroid inducedosteoporosis, senile osteoporosis or postmenopausal osteoporosis, aswell as osteomalacia and renal osteodystrophy. The treatment may betransdermal, oral or parenteral. The compounds may be present in acomposition in an amount from about 0.1 μg/gm to about 50 μg/gm of thecomposition, and may be administered in dosages of from about 0.9 μg/dayto about 50 μg/day.

[0021] The compounds of the invention are also especially suited fortreatment and i prophylaxis of human disorders which are characterizedby an imbalance in the immune system, e.g. in autoimmune diseases,including multiple sclerosis, diabetes mellitus, host versus graftreaction, and rejection of transplants; and additionally for thetreatment of inflammatory diseases, such as rheumatoid arthritis andasthma, as well as the improvement of bone fracture healing and improvedbone grafts. Acne, alopecia, skin conditions such as dry skin (lack ofdermal hydration), undue skin slackness (insufficient skin firmness),insufficient sebum secretion and wrinkles, and hypertension are otherconditions which may be treated with the compounds of the invention.

[0022] The above compounds are also characterized by high celldifferentiation activity. Thus, these compounds also provide therapeuticagents for the treatment of psoriasis, or as an anti-cancer agent,especially against leukemia, colon cancer, breast cancer and prostatecancer. The compounds may be present in a composition to treat psoriasisin an amount from about 0.01 μg/gm to about 100 μg/gm of thecomposition, and may be administered topically, transdermally, orally orparenterally in dosages of from about 0.01 μg/day to about 100 μg/day.

[0023] This invention also provides novel intermediate compounds formedduring the synthesis of the end products.

[0024] This invention also provides a novel synthesis for the productionof the end products of structure I.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 is a graph illustrating the relative activity of a mixtureof 2α and 2β-methyl-19-nor-20S-1α,25-dihydroxyvitamin D₃, a mixture of2α and 2β-methyl-19-nor-1α,25-dihydroxyvitamin D₃ and1α,25-dihydroxyvitamin D₃ to compete for binding of [³H]-1,25-(OH)₂-D₃to the pig intestinal nuclear vitamin D receptor;

[0026]FIG. 2 is a graph illustrating the percent HL-60 celldifferentiation as a function of the concentration of a mixture of 2αand 2β-methyl-19-nor-20S-1α,25-dihydroxyvitamin D₃, a mixture of 2α and2β-methyl-19-nor-1,25-dihydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃;

[0027]FIG. 3 if a graph similar to FIG. 1 except illustrating therelative activity of the individual compounds 2α and2β-methyl-19-nor-20S-1α,25-dihydroxyvitamin D₃,2α and2,2-methyl-19-nor-1α,25-dihydroxyvitamin D₃ and 1α,25-dihydroxyvitaminD₃ to compete for binding of [³H]-1,25-(OH)₂-D₃ to the vitamin D pigintestinal nuclear receptor;

[0028]FIG. 4 is a graph similar to FIG. 2 except illustrating thepercent HL-60 cell differentiation as a function of the concentration ofthe individual compounds 2α and2β-methyl-19-nor-20S-1α,25-dihydroxyvitamin D₃, 2α and2β-methyl-19-nor-1α,25-dihydroxyvitamin D₃ and 1α,25-dihydroxyvitaminD₃;

[0029]FIG. 5 is a graph illustrating the relative activity of theindividual compounds 2α and2β-hydroxymethyl-19-nor-20S-1α,25-dihydroxyvitamin D₃, 2α and2β-hydroxymethyl-19-nor-1α,25-dihydroxyvitamin D₃ and1α,25-dihydroxyvitamin D₃ to complete for binding of [³H]-1,25-(OH)₂-D₃to the vitamin D pig intestinal nuclear receptor; and

[0030]FIG. 6 is a graph illustrating the percent HL-60 celldifferentiation as a function of the concentration of the individualcompounds 2α and 2β-hydroxymethyl-19-nor-20S-1α,25-dihydroxyvitamin D₃,2α and 2β-hydroxymethyl-19-nor-1α,25-dihydroxyvitamin D₃ and1α,25-dihydroxyvitamin D₃.

DETAILED DESCRIPTION OF THE INVENTION

[0031] As used in the description and in the claims, the term“hydroxy-protecting group” signifies any group commonly used for thetemporary protection of hydroxy functions, such as for example,alkoxycarbonyl, acyl, alkylsilyl or alkylarylsilyl groups (hereinafterreferred to simply as “silyl” groups), and alkoxyalkyl groups.Alkoxycarbonyl protecting groups are alkyl-O—CO— groupings such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl,butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl,benzyloxycarbonyl or allyloxycarbonyl. The term “acyl” signifies analkanoyl group of 1 to 6 carbons, in all of its isomeric forms, or acarboxyalkanoyl group of 1 to 6 carbons, such as an oxalyl, malonyl,succinyl, glutaryl group, or an aromatic acyl group such as benzoyl, ora halo, nitro or alkyl substituted benzoyl group. The word “alkyl” asused in the description or the claims, denotes a straight-chain orbranched alkyl radical of 1 to 10 carbons, in all its isomeric forms.Alkoxyalkyl protecting groups are groupings such as methoxymethyl,ethoxymethyl, methoxyethoxymethyl, or tetrahydrofuranyl andtetrahydropyranyl. Preferred silyl-protecting groups are trimethylsilyl,triethylsilyl, t-butyldimethylsilyl, dibutylmethylsilyl,diphenylmethylsilyl, phenyldimethylsilyl, diphenyl-t-butylsilyl andanalogous alkylated silyl radicals. The term “aryl” specifies a phenyl-,or an alkyl-, nitro- or halo-substituted phenyl group.

[0032] A “protected hydroxy” group is a hydroxy group derivatised orprotected by any of the above groups commonly used for the temporary orpermanent protection of hydroxy functions, e.g. the silyl, alkoxyalkyl,acyl or alkoxycarbonyl groups, as previously defined. The terms“hydroxyalkyl”, “deuteroalkyl” and “fluoroalkyl” refer to an alkylradical substituted by one or more hydroxy, deuterium or fluoro groupsrespectively.

[0033] It should be noted in this description that the term “24-homo”refers to the addition of one methylene group and the term “24-dihomo”refers to the addition of two methylene groups at the carbon 24 positionin the side chain. Likewise, the term “trihomo” refers to the additionof three methylene groups. Also, the term “26,27-dimethyl” refers to theaddition of a methyl group at the carbon 26 and 27 positions so that forexample R³ and R⁴ are ethyl groups. Likewise, the term “26,27-diethyl”refers to the addition of an ethyl group at the 26 and 27 positions sothat R³ and R⁴ are propyl groups.

[0034] In the following lists of compounds, the particular substituentattached at the carbon 2 position should be added to the nomenclature.For example, if a methyl group is the alkyl substituent, the term“2-methyl” should precede each of the named compounds. If an ethyl groupis the alkyl substituent, the term “2-ethyl” should precede each of thenamed compounds, and so on. In addition, if the methyl group attached atthe carbon 20 position is in its epi or unnatural configuration, theterm “20(S)” or “20-epi” should be included in each of the followingnamed compounds. Also, if the side chain contains an oxygen atomsubstituted at any of positions 20, 22 or 23, the term “20-oxa”,422-oxa” or “23-oxa”, respectively, should be added to the namedcompound. The named compounds could also be of the vitamin D₂ type ifdesired.

[0035] Specific and preferred examples of the 2-alkyl-compounds ofstructure I when the side chain is unsaturated are:

[0036] 19-nor-24-homo-1,25-dihydroxy-22,23-dehydrovitamin D₃;

[0037] 19-nor-24-dihomo-1,25-dihydroxy-22,23-dehydrovitarin D₃;

[0038] 19-nor-24-trihomo-1,25-dihydroxy-22,23-dehydrovitamin D₃;

[0039] 19-nor-26,27-dimethyl-24-homo-1,25-dihydroxy-22,23-dehydrovitaminD₃;

[0040]19-nor-26,27-dimethyl-24-dihomo-1,25-dihydroxy-22,23-dehydrovitamin D₃;

[0041]19-nor-26,27-dimethyl-24-trihomo-1,25-dihydroxy-22,23-dehydrovitamin D₃;

[0042] 19-nor-26,27-diethyl-24-homo-1,25-dihydroxy-22,23-dehydrovitaminD₃;

[0043]19-nor-26,27-diethyl-24-dihomo-1,25-dihydroxy-22,23-dehydrovitamin D₃;

[0044]19-nor-2627-diethyl-24-trihomo-1,25-dihydroxy-22,23-dehydrovitarin D₃;

[0045]19-nor-26,27-dipropoyl-24-homo-1,25-dihydroxy-22,23-dehydrovitamin D₃;

[0046]19-nor-26,27-dipropyl-24-dihomo-1,25-dihydroxy-22,23-dehydrovitamin D₃;and

[0047]19-nor-26,27-dipropyl-24-trihomo-1,25-dihydroxy-22,23-dehydrovitamin D₃.

[0048] With respect to the above unsaturated compounds, it should benoted that the double bond located between the 22 and 23 carbon atoms inthe side chain may be in either the (E) or (Z) configuration.Accordingly, depending upon the configuration, the term “22,23(E)” or“22,23(Z)” should be included in each of the above named compounds.Also, it is common to designate the double bond located between the 22and 23 carbon atoms with the designation “Δ²²”. Thus, for example, thefirst named compound above could also be written as19-nor-24-homo-22,23(E)-Δ²²-1,25-(OH)₂D₃ where the double bond is in the(E) configuration. Similarly, if the methyl group attached at carbon 20is in the unnatural configuration, this compound could be written as19-nor-20(S)-24-homo-22,23)-Δ²²-1,25-(OH)₂D₃.

[0049] Specific and preferred examples of the 2-alkyl-compounds ofstructure I when the side chain is saturated are:

[0050] 19-nor-24-homo-1,25-dihydroxyvitamin D₃;

[0051] 19-nor-24-dihomo-1,25-dihydroxyvitamin D₃;

[0052] 19-nor-24-trihomo-1,25-dihydroxyvitamin D₃;

[0053] 19-nor-26,27-dimethyl-24-homo-1,25-dihydroxyvitamin D₃;

[0054] 19-nor-26,27-dimethyl-24-dihomo-1,25-dihydroxyvitamin D₃;

[0055] 19-nor-26,27-dimethyl-24-trihomo-1,25-dihydroxyvitamin D₃;

[0056] 19-nor-26,27-diethyl-24-homo-1,25-dihydroxyvitamin D₃;

[0057] 19-nor-26,27-diethyl-24-dihomo-1,25-dihydroxyvitamin D₃;

[0058] 19-nor-26,27-diethyl-24-trihomo-1,25-dihydroxyvitamin D₃;

[0059] 19-nor-26,27-dipropyl-24-homo-1,25-dihydroxyvitamin D₃;

[0060] 19-nor-26,27-dipropyl-24-dihomo-1,25-dihydroxyvitamin D₃; and

[0061] 19-nor-26,27-dipropyl-24-trihomo-1,25-dihydroxyvitamin D₃.

[0062] As noted previously, the above saturated side chain compoundsshould have the appropriate 2-alkyl substituent and/or carbon 20configuration added to the nomenclature. For example, particularlypreferred compounds are:

[0063] 19-nor-26,27-dimethyl-20(S)-2α-methyl-1α,25-dihydroxyvitamin D₃;which can also be written as19-nor-26,27-dihomo-20(S)-2α-methyl-1α,25-dihydroxyvitamin D₃;

[0064] 19-nor-26,27-dimethyl-20(S)-2β-methyl-1α,25-dihydroxyvitamin D₃;which can also be written as19-nor-26,27-dihomo-20(S)-2β-methyl-1α,25-dihydroxyvitamin D₃;

[0065] 19-nor-26,27-dimethylene-20(S)-2α-methyl-1α,25-dihydroxyvitaminD₃; and

[0066] 19-nor-26,27-dimethylene-20(S)-2β-methyl-1α,25-dihydroxyvitaminD₃.

[0067] The preparation of 1α-hydroxy-2-alkyl-19-nor-vitamin D compounds,particularly p-hydroxy-2-methyl-19-nor-vitamin D compounds, having thebasic structure I can be accomplished by a common general method, i.e.the condensation of a bicyclic Windaus-Grundmann type ketone II with theallylic phosphine oxide III to the corresponding2-methylene-19-nor-vitamin D analogs IV followed by a selectivereduction of the exomethylene group at C-2 in the latter compounds:

[0068] In the structures II, III, and IV groups Y, and Y₂ and Rrepresent groups defined above; Y, and Y₂ are preferablyhydroxy-protecting groups, it being also understood that anyfunctionalities in R that might be sensitive, or that interfere with thecondensation reaction, be suitable protected as is well-known in theart. The process shown above represents an application of the convergentsynthesis concept, which has been applied effectively for thepreparation of vitamin D compounds [e.g. Lythgoe et al., J. Chem. Soc.Perkin Trans. I, 590 (1978); Lythgoe, Chem. Soc. Rev. 9, 449 (1983); Tohet al., J. Org. Chem. 4.8 1414 (1983); Baggiolini et al., J. Org. Chem.51, 3098 (1986); Sardina et al., J. Org. Chem. 51 1264 (1986); J. Org.Chem. Li, 1269 (1986); DeLuca et al., U.S. Pat. No. 5,086,191; DeLuca etal., U.S. Pat. No. 5,536,713].

[0069] Hydrindanones of the general structure II are known, or can beprepared by known methods. Specific important examples of such knownbicyclic ketones are the structures with the side chains (a), (b), (c)and (d) described above, i.e. 25-hydroxy Grundmann's ketone (f)[Baggiolini et al., J. Org. Chem, 51,3098 (1986)]; Grundmann's ketone(g) [Inhoffen et al., Chem. Ber. 90 664 (1957)]; 25-hydroxy Windausketone (h) [Baggiolini et al., J. Org. Chem., 51, 3098 (1986)] andWindaus ketone (i) [Windaus et al., Ann., 524,297 (1936)]:

[0070] For the preparation of the required phosphine oxides of generalstructure III, a new synthetic route has been developed starting frommethyl quinicate derivative 1, easily obtained from commercial(1R,3R,4S,5R)-(−)-quinic acid as described by Perlman et al.,Tetrahedron Lett. 32 7663 (1991) and DeLuca et al., U.S. Pat. No.5,086,191. The overall process of transformation of the starting methylester 1 into the desired A-ring synthons, is summarized by the SCHEME I.Thus, the secondary 4-hydroxyl group of 1 was oxidized with RuO₄ (acatalytic method with RuCl₃ and NaIO₄ as co-oxidant). Use of such astrong oxidant was necessary for an effective oxidation process of thisvery hindered hydroxyl. However, other more commonly used oxidants canalso be applied (e.g. pyridinium dichromate), although the reactionsusually require much longer time for completion. Second step of thesynthesis comprises the Wittig reaction of the sterically hindered4-keto compound 2 with ylide prepared from methyltriphenylphosphoniumbromide and n-butyllithium. Other bases can be also used for thegeneration of the reactive methylenephosphorane, like t-BuOK, NaNH₂,NaH, K/HMPT, NaN(TMS)₂, etc. For the preparation of the 4-methylenecompound 3 some described modifications of the Wittig process can beused, e.g. reaction of 2 with activated methylenetriphenyl-phosphorane[Corey et al., Tetrahedron Lett. 26, 555 (1985)]. Alternatively, othermethods widely used for methylenation of unreactive ketones can beapplied, e.g. Wittig-Homer reaction with the PO-ylid obtained frommethyldiphenylphosphine oxide upon deprotonation with n-butyllithium[Schosse et al., Chimia 3, 197 (1976)], or reaction of ketone withsodium methylsulfinate [Corey et al., J. Org. Chem. 2, 1128 (1963)] andpotassium methylsulfinate [Greene et al., Tetrahedron Lett. 3755(1976)]. Reduction of the ester with lithium aluminum hydride or othersuitable reducing agent (e.g. DIBALH) provided the diol 4 which wassubsequently oxidized by sodium periodate to the cyclohexanonederivative 5. The next step of the process comprises the Petersonreaction of the ketone 5 with methyl(trimethylsilyl)acetate. Theresulting allylic ester 6 was treated with diisobutylaluminum hydrideand the formed allylic alcohol 7 was in turn transformed to the desiredA-ring phosphine oxide 8. Conversion of 7 to 8 involved 3 steps, namely,in situ tosylation with n-butyllithium and p-toluenesulfonyl chloride,followed by reaction with diphenylphosphine lithium salt and oxidationwith hydrogen peroxide.

[0071] Several 2-methylene-19-nor-vitamin D compounds of the generalstructure IV may be synthesized using the A-ring synthon 8 and theappropriate Windaus-Grundmann ketone II having the desired side chainstructure. Thus, for example, Wittig-Horner coupling of the lithiumphosphinoxy carbanion generated from 8 and n-butyllithium with theprotected 25-hydroxy Grundmann's ketone 9 prepared according topublished procedure [Sicinski et al., J. Med. Chem. 37, 3730 (1994)]gave the expected protected vitamin compound 10. This, afterdeprotection with AG 50W-X4 cation exchange resin afforded1α,25-dihydroxy-2-methylene-19-nor-vitamin D₃ (11).

[0072] The final step of the process was the selective homogeneouscatalytic hydrogenation of the exomethylene unit at carbon 2 in thevitamin 11 performed efficiently in the presence oftris(triphenylphosphine)rhodium(I) chloride [Wilkinson's catalyst,(Ph₃P)₃RhCl]. Such reduction conditions allowed to reduce only C(2)=CH₂unit leaving C(5)-C(8) butadiene moiety unaffected. The isolatedmaterial is an epimeric mixture (ca. 1:1) of 2-methyl-19-nor-vitamins 12and 13 differing in configuration at C-2. The mixture can be usedwithout separation or, if desired, the individual 2α- and 2β-isomers canbe separated by an efficient HPLC system.

[0073] A similar chemoselectivity was also observed in hydroborationreactions to synthesize 2-hydroxymethyl derivatives 20 and 21 (seeScheme III). For this purpose, 9-borabicyclo(3.3.1)nonane (9-BBN) wasused as a reagent and reaction conditions analogous as those used byOkamura for hydroboration of simple vitamin D compounds. See J. Org.Chem. 1978,43, 1653-1656 and J. Org. Chem. 1977,42,2284-2291. Since thisliterature precedent concerned hydroboration of 1-desoxy compounds,namely, (5E)- and (5Z)-isomers of vitamin D₂ and D₃, the process wasfirst tested using 1α,25-(OH)₂D₃ as a model compound. The formedorganoborane intermediate was subsequently oxidized with basic hydrogenperoxide. Such hydroboration-oxidation conditions allowed the exclusivehydroxylation of the C(2)=CH₂ unit in the vitamin 11, leaving theintercyclic C(5)=C(6)-C(7)=C(8) diene moiety unaffected. The isolatedepimeric mixture of 2-hydroxymethyl derivatives 20 and 21 (ca. 1:2, 35%yield) was purified and separated by straight- and reversed-phase HPLC.

[0074] The C-20 epimerization was accomplished by the analogous couplingof the phosphine oxide 8 with protected 20(S)-25-hydroxy Grundmann'sketone 15 (SCHEME II) and provided 19-nor-vitamin 16 which afterhydrolysis of the hydroxy-protecting groups gave20(S)-1α,25-dihydroxy-2-methylene-19-nor-vitamin D₃ (17). Hydrogenationof 17 using Wilkinson's catalyst provided the expected mixture of the2-methyl-19-nor-vitamin D analogs 18 and 19. Subsequent hydroborationwith 9-BBN yielded 20(S)-2-hydroxymethyl derivatives 22 and 23 (seeScheme III).

[0075] As noted above, other 2-methyl-19-nor-vitamin D analogs may besynthesized by the method disclosed herein. For example,1α-hydroxy-2-methylene-19-nor-vitamin D₃ can be obtained by providingthe Grundmann's ketone (g); subsequent reduction of the A-ringexomethylene group in the formed compound can give the correspondingepimeric mixture of 1α-hydroxy-2-methyl-19-nor-vitamin D₃ compounds.

[0076] A number of oxa-analogs of vitamin D₃ and their synthesis arealso known. For example, 20-oxa analogs are described in N. Kubodera atal, Chem. Pharm. Bull., 34, 2286 (1986), and Abe et al, FEBS Lett. 222,58, 1987. Several 22-oxa analogs are described in E. Murayama et al,Chem. Pharm. Bull., 34,4410 (1986), Abe et al, FEBS Lett., 226, 58(1987), PCT International Application No. WO 90/09991 and EuropeanPatent Application, publication number 184 112, and a 23-oxa analog isdescribed in European Patent Application, publication number 78704, aswell as U.S. Pat. No. 4,772,433.

[0077] This invention is described by the following illustrativeexamples. In these. examples specific products identified by Arabicnumerals (e.g. 1, 2, 3, etc) refer to the specific structures soidentified in the preceding description and in the SCHEME I and SCHEMEE.

EXAMPLE 1

[0078] Preparation of 1α,25-dihydroxy-2α- and1α,25-dihydroxy-2β-methyl-19-nor-vitamin D₃ (12 and 13).

[0079] Referring first to SCHEME I the starting methyl quinicatederivative 1 was obtained from commercial (−)-quinic acid as describedpreviously [Perlman et al., Tetrahedron Lett. 32, 7663 (1991) and DeLucaet al., U.S. Pat. No. 5,086,191]. 1: mp. 82-82.5° C. (from hexane), ¹HNMR (CDCl₃) δ 0.098, 0.110, 0.142, and 0.159 (each 3H, each s, 4×SiCH₃), 0.896 and 0.911 (9H and 9H, each s, 2× Si-t-Bu), 1.820 (1H, dd,J=13.1, 10.3 Hz), 2.02 (1H, ddd, J=14.3, 4.3, 2.4 Hz), 2.09 (1H, dd,J=14.3, 2.8 Hz), 2.19 (1H, ddd, J=13.1, 4.4, 2.4 Hz), 2.31 (1H, d, J=2.8Hz, OH), 3.42 (1H, m; after D₂O dd, J=8.6, 2.6 Hz), 3.77 (3H, s), 4.12(1H, m), 4.37 (1H, m), 4.53 (1H, br s, OH).

[0080] (a) Oxidation of 4-hydroxy Group in Methyl Quinicate Derivative1.

[0081](3R,5R)-3,5-Bis[(tert-butyldimethylsilyl)oxy]-1-hydroxy-4-oxocyclohexanecarboxylicAcid Methyl Ester (2). To a stirred mixture of ruthenium(III) chloridehydrate (434 mg, 2.1 mmol) and sodium periodate (10.8 g, 50.6 mmol) inwater (42 mL) was added a solution of methyl quinicate 1 (6.09 g, 14mmol) in CCl₄/CH₃CN (1:1, 64 mL). Vigorous stirring was continued for 8h. Few drops of 2-propanol were added, the mixture was poured into waterand extracted with chloroform. The organic extracts were combined,washed with water, dried (MgSO₄) and evaporated to give a dark oilyresidue (ca. 5 g) which was purified by flash chromatography. Elutionwith hexane/ethyl acetate (8:2) gave pure, oily 4-ketone 2 (3.4 g, 56%):¹H NMR (CDCl₃) δ 0.054, 0.091, 0.127, and 0.132 (each 3H, each s, 4×SiCH₃), 0.908 and 0.913 (9H and 9H, each s, 2× Si-t-Bu), 2.22 (1H, dd,J=13.2, 11.7 Hz), 2.28 (1H, dt, J=14.9, 3.6 Hz), 2.37 (1H, dd, J=14.9,3.2 Hz), 2.55 (1H, ddd, J=13.2, 6.4, 3.4 Hz), 3.79 (3H, s), 4.41 (1H, t,J˜3.5 Hz), 4.64 (1H, s, OH), 5.04 (1H, dd, J=11.7, 6.4 Hz); MS m/z(relative intensity) no M⁺, 375 (M⁺-t-Bu, 32), 357 (M⁺-t-Bu-H₂O, 47),243 (31), 225 (57), 73 (100).

[0082] (b) Wittig Reaction of the 4-ketone 2.

[0083](3R,5R)-3,5-Bis[(tert-butyldimethylsilyl)oxy]-1-hydroxy-4-methylenecyclohexanecarboxylicAcid Methyl Ester (3). To the methyltriphenylphoshonium bromide (2.813g, 7.88 mmol) in anhydrous THF (32 mL) at 0° C. was added dropwisen-BuLi (2.5 M in hexanes, 6.0 mL, 15 mmol) under argon with stirring.Another portion of MePh₃P⁺Br (2.813 g, 7.88 mmol) was then added and thesolution was stirred at 0° C. for 10 min and at room temperature for 40min. The orange-red mixture was again cooled to 0° C. and a solution of4-ketone 2 (1.558 g, 3.6 mmol) in anhydrous THF (16+2 mL) was syphonedto reaction flask during 20 min. The reaction mixture was stirred at 0°C. for 1 h and at room temperature for 3 h. The mixture was thencarefully poured into brine cont. 1% HCl and extracted with ethylacetate and benzene. The combined organic extracts were washed withdiluted NaHCO₃ and brine, dried (MgSO₄) and evaporated to give an orangeoily residue (ca. 2.6 g) which was purified by flash chromatography.Elution with hexane/ethyl acetate (9:1) gave pure 4-methylene compound 3as a colorless oil (368 mg, 24%): ¹H NMR (CDCl₃) δ 0.078, 0.083, 0.092,and 0.115 (each 3H, each s, 4× SiCH₃), 0.889 and 0.920 (9H and 9H, eachs, 2× Si-t-Bu), 1.811 (1H, dd, J=12.6, 11.2 Hz), 2.10 (2H, m), 2.31 (1H,dd, J=12.6, 5.1 Hz), 3.76 (3H, s), 4.69 (1H, t, J=3.1 Hz), 4.78 (1H, m),4.96 (2H, m; after D₂OH, br s), 5.17 (1H, t, J=1.9 Hz); MS m/z (relativeintensity) no M⁺, 373 (M⁺-t-Bu, 57), 355 (M⁺-t-Bu-H₂O, 13), 341 (19),313 (25), 241 (33), 223 (37), 209 (56), 73 (100).

[0084] (c) Reduction of Ester Group in the 4-methylene Compound 3.

[0085][(3′R,5′R)-3′,5′-Bis[(tert-butyldimethylsilyl)oxy]-1-hydroxy-4′-methylenecyclohexyl]methanol(4). (i) To a stirred solution of the ester 3 (90 mg, 0.21 mmol) inanhydrous THF (8 mL) lithium aluminum hydride (60 mg, 1.6 mmol) wasadded at 0° C. under argon. The cooling bath was removed after 1 h andthe stirring was continued at 6° C. for 12 h and at room temperature for6 h. The excess of the reagent was decomposed with saturated aq. Na₂SO₄,and the mixture was extracted with ethyl acetate and ether, dried(MgSO₄) and evaporated. Flash chromatography of the residue withhexanelethyl acetate (9:1) afforded unreacted substrate (12 mg) and apure, crystalline diol 4 (35 mg, 48% based on recovered ester 3): ¹H NMR(CDCl₃+D₂O) δ 0.079, 0.091, 0.100, and 0.121 (each 3H, each s, 4×SiCH₃), 0.895 and 0.927 (9H and 9H, each s, 2× Si-t-Bu), 1.339 (1H, t,J˜12 Hz), 1.510 (1H, dd, J=14.3, 2.7 Hz), 2.10 (2H, m), 3.29 and 3.40(1H and 1H, each d, J=11.0 Hz), 4.66 (1H, t, J˜2.8 Hz), 4.78 (1H, m),4.92 (1H, t, J=1.7 Hz), 5.13 (1H, t, J=2.0 Hz); MS m/z (relativeintensity) no M⁺, 345 (M⁺-t-Bu, 8), 327 (M⁺-t-Bu-H₂O, 22), 213 (28), 195(11), 73 (100). (ii) Diisobutylaluminum hydride (1.5 M in toluene, 2.0mL, 3 mmol) was added to a solution of the ester 3 (215 mg, 0.5 mmol) inanhydrous ether (3 mL) at −78° C. under argon. The mixture was stirredat −78° C. for 3 h and at −24° C. for 1.5 h, diluted with ether (10 mL)and quenched by the slow addition of 2N potassium sodium tartrate. Thesolution was warmed to room temperature and stirred for 15 min, thenpoured into brine and extracted with ethyl acetate and ether. Theorganic extracts were combined, washed with diluted (ca. 1%) HCl, andbrine, dried (MgSO₄) and evaporated. The crystalline residue waspurified by flash chromatography. Elution with hexane/ethyl acetate(9:1) gave crystalline diol 4 (43 mg, 24%).

[0086] (d) Cleavage of the Vicinal Diol 4.

[0087](3R,5R)-3,5-Bis[(tert-butyldimethylsilyl)oxy]-4-methylenecyclohexanone(5). Sodium periodate saturated water (2.2 mL) was added to a solutionof the diol 4 (146 mg, 0.36 mmol) in methanol (9 mL) at 0° C. Thesolution was stirred at 0° C. for 1 h, poured into brine and extractedwith ether and benzene. The organic extracts were combined, washed withbrine, dried (MgSO₄) and evaporated. An oily residue was dissolved inhexane (1 mL) and applied on a silica Sep-Pak cartridge. Pure4-methylenecyclohexanone derivative 5 (110 mg, 82%) was eluted withhexane/ethyl acetate (95:5) as a colorless oil: ¹H NMR (CDCl₃) δ 0.050and 0.069 (6H and 6H, each s, 4× SiCH₃), 0.881 (18H, s, 2× Si-t-Bu),2.45 (2H, ddd, J=14.2, 6.9, 1.4 Hz), 2.64 (2H, ddd, J=14.2, 4.6, 1.4Hz), 4.69 (2H, dd, J=6.9, 4.6 Hz), 5.16 (2H, s); MS m/z (relativeintensity) no M⁺, 355 (M⁺-Me, 3), 313 (M⁺-t-Bu, 100), 73 (76).

[0088] (e) Preparation of the Allylic Ester 6.

[0089][(3′R,5′R)-3′,5′-Bis[(tert-butyldimethylsilyl)oxy]-4′-methylenecyclohexylidene]aceticAcid Methyl Ester (6). To a solution of diisopropylamine (37 μL, 0.28mmol) in anhydrous THEF (200 μL) was added n-BuLi (2.5 M in hexanes, 113μL, 0.28 mmol) under argon at −78° C. with stirring, andmethyl(trimethylsilyl)acetate (46 μL, 0.28 mmol) was then added. After15 min, the keto compound 5 (49 mg, 0.132 mmol) in anhydrous THF (200+80μL) was added dropwise. The solution was stirred at −78° C. for 2 h andthe reaction mixture was quenched with saturated NH₄Cl, poured intobrine and extracted with ether and benzene. The combined organicextracts were washed with brine, dried (MgSO₄) and evaporated. Theresidue was dissolved in hexane (1 mL) and applied on a silica Sep-Pakcartridge. Elution with hexane and hexane/ethyl acetate (98:2) gave apure allylic ester 6 (50 mg, 89%) as a colorless oil: ¹H NMR (CDCl₃) δ0.039, 0.064, and 0.076 (6H, 3H, and 3H, each s, 4× SiCH₃), 0.864 and0.884 (9H and 9H, each s, 2× Si-t-Bu), 2.26 (1H, dd, J=12.8, 7.4 Hz),2.47 (1H, dd, J=12.8, 4.2 Hz), 2.98 (1H, dd, J=13.3, 4.0 Hz), 3.06 (1H,dd, 3=13.3, 6.6 Hz), 3.69 (3H, s), 4.48 (2H, m), 4.99 (2H, s), 5.74 (1H,s); MS m/z (relative intensity) 426 (M⁺, 2), 411 (M⁺-Me, 4), 369(M⁺-t-Bu, 100), 263 (69).

[0090] (f) Reduction of the Allylic Ester 6.

[0091]2-[(3′R,5′R)-3′,5′-Bis[(tert-butyldimethylsilyl)oxy]-4′-methylenecyclohexylidene]ethanol (7). Diisobutylaluminum hydride (1.5 M in toluene, 1.6 mL, 2.4mmol) was slowly added to a stirred solution of the allylic ester 6 (143mg, 0.33 mmol) in toluene/methylene chloride (2:1, 5.7 mL) at −78° C.under argon. Stirring was continued at −78° C. for 1 h and at −46° C.(cyclohexanone/dry ice bath) for 25 min. The mixture was quenched by theslow addition of potassium sodium tartrate (2N, 3 mL), aq. HCl (2N, 3mL) and H₂O (12 mL), and then diluted with methylene chloride (12 mL)and extracted with ether and benzene. The organic extracts werecombined, washed with diluted (ca. 1%) HCl, and brine, dried (MgSO₄) andevaporated. The residue was purified by flash chromatography. Elutionwith hexane/ethyl acetate (9:1) gave crystalline allylic alcohol 7 (130mg, 97%): ¹H NMR (CDCl₃) δ 0.038, 0.050, and 0.075 (3H, 3H, and 6H, eachs, 4× SiCH₃), 0.876 and 0.904 (9H and 9H, each s, 2× Si-t-Bu), 2.12 (1H,dd, J=12.3, 8.8 Hz), 2.23 (1H, dd, J=13.3, 2.7 Hz), 2.45 (1H, dd,J=12.3, 4.8 Hz), 2.51 (1H, dd, J=13.3, 5.4 Hz), 4.04 (1H, m; after D₂Odd, J=12.0, 7.0 Hz), 4.17 (1H, m; after D₂O dd, J=12.0, 7.4 Hz), 4.38(1H, m), 4.49 (1H, m), 4.95 (1H, br s), 5.05 (1H, t, J=1.7 Hz), 5.69(1H, t, J=7.2 Hz); MS m/z (relative intensity) 398 (M⁺, 2), 383 (M⁺-Me,2), 365 (M⁺-Me-H₂O, 4), 341 (M⁺-t-Bu, 78), 323 (M⁺-t-Bu-H₂O, 10), 73(100); exact mass calcd for C₂₇H₄₄O₃ 416.3290, found 416.3279.

[0092] (g) Conversion of the Allylic Alcohol 7 into Phosphine Oxide 8.

[0093][2-[(3′R,5′R)-3′,5′-Bis[(tert-butyldimethylsilyl)oxy]-4′-methylenecyclohexylidene]ethyl]diphenylphosphineOxide (8). To the allylic alcohol 7 (105 mg, 0.263 mmol) in anhydrousTHF (2.4 mL) was added n-BuLi (2.5 M in hexanes, 105 μL, 0.263 mmol)under-argon at 0° C. Freshly recrystallized tosyl chloride (50.4 mg,0.264 mmol) was dissolved in anhydrous THE (480 μL)and added to theallylic alcohol-BuLi solution. The mixture was stirred at 0° C. for 5min and set aside at 0° C. In another dry flask with air replaced byargon, n-BuLi (2.5 M in hexanes, 210 μL, 0.525 mmol) was added to Ph₂PH(93 μL, 0.534 mmol) in anhydrous THF (750 μL) at 0° C. with stirring.The red solution was syphoned under argon pressure to the solution oftosylate until the orange color persisted (ca. 112 of the solution wasadded). The resulting mixture was stirred an additional 30 min at 0° C.,and quenched by addition of H₂O (30 μl). Solvents were evaporated underreduced pressure and the residue was redissolved in methylene chloride(2.4 mL) and stirred with 10% H₂O₂ at 0° C. for 1 h. The organic layerwas separated, washed with cold aq. sodium sulfite and H₂O, dried(MgSO₄) and evaporated. The residue was subjected to flashchromatography. Elution with benzenelethyl acetate (6:4) gavesemicrystalline phosphine oxide 8 (134 mg, 87%): ¹H NMR (CDCl₃) δ 0.002,0.011, and 0.019 (3H, 3H, and 6H, each s, 4× SiCH₃), 0.855 and 0.860 (9Hand 9H, each s, 2× Si-t-Bu), 2.0-2.1 (3H, br m), 2.34 (1H, m), 3.08 (1H,m), 3.19 (1H, m), 4.34 (2H, m), 4.90 and 4.94 (1H and 1H, each s,), 5.35(1H, ˜q, J=7.4 Hz), 7.46 (4H, m), 7.52 (2H, m), 7.72 (4H, m); MS-m/z(relative intensity) no M⁺, 581 (M⁺-1,1), 567 (M⁺-Me, 3), 525 (M⁺-t-Bu,100), 450 (10), 393 (48).

[0094] (h) Wittig-Homer Coupling of Protected 25-hydroxy Grundmann'sKetone 9 with the Phosphine Oxide 8.

[0095] 1α,25-Dihydroxy-2-methylene-19-nor-vitamin D₃ (11). To a solutionof phosphine oxide 8 (33.1 mg, 56.8 μmol) in anhydrous THB (450 μL) at0° C. was slowly added n-BuLi (2.5 M in hexanes, 23 μL, 57.5 μmol) underargon with stirring. The solution turned deep orange. The mixture wascooled to −78° C. and a precooled (−78° C.) solution of protectedhydroxy ketone 9 (9.0 mg, 22.8 μmol), prepared according to publishedprocedure [Sicinski et al., S. Med. Chem. 37,3730 (1994)], in anhydrousTHF (200+100 μL) was slowly added. The mixture was stirred under argonat −78° C. for 1 h and at 0° C. for 18 h. Ethyl acetate was added, andthe organic phase was washed with brine, dried (MgSO₄) and evaporated.The residue was dissolved in hexane and applied on a silica Sep-Pakcartridge, and washed with hexane/ethyl acetate (99:1, 20 mL) to give19-nor-vitamin derivative 10 (13.5 mg, 78%). The Sep-Pak was then washedwith hexane/ethyl acetate (96:4, 10 mL) to recover some unchangedC,D-ring ketone 9 (2 mg), and with ethyl acetate (10 mL) to recoverdiphenylphosphine oxide (20 mg). For analytical purpose a sample ofprotected vitamin 10 was further purified by HPLC (6.2 mm×25 cmZorbax-Sil column, 4 mL/min) using hexane/ethyl acetate (99.9:0.1)solvent system. Pure compound 10 was eluted at R_(V) 26 mL as acolorless oil: UV (in hexane) λ_(max) 244, 253, 263 nm; ¹H NMR (CDCl₃) δ0.025, 0.049, 0.066, and 0.080 (each 3H, each s, 4× SiCH₃), 0.546 (3H,s, 18-H₃), 0.565 (6H, q, J=7.9 Hz, 3× SiCH₂), 0.864 and 0.896 (9H and9H, each s, 2× Si-t-Bu), 0.931 (3H, d, J=6.0 Hz, 21-H₃), 0.947 (9H, t,J=7.9 Hz, 3× SiCH₂CH₃), 1.188 (6H, s,26- and 27-H₃), 2.00 (2H, m), 2.18(1H, dd, J=12.5, 8.5 Hz, 4β-1H), 2.33 (1H, dd, J=13.1, 2.9 Hz, 10β-H),2.46 (1H, dd, J=12.5, 4.5 Hz, 4α-H), 2.52 (1H, dd, J=13.1, 5.8 Hz,10α-H), 2.82 (1H, br d, J=12 Hz, 90-H), 4.43 (2H, m, 1β- and 3α-H), 4.92and 4.97(1H and 1H, each s, ═CH₂), 5.84 and 6.22 (1H and 1H, each d,J=11.0 Hz, 7- and 6-H); MS m/z (relative intensity) 758 (M⁺, 17), 729(M⁺-Et, 6), 701 (M⁺-t-Bu, 4), 626 (100), 494 (23), 366 (50),73 (92).

[0096] Protected vitamin 10 (4.3 mg) was dissolved in benzene (150 μL)and the resin (AG 50W-X4, 60 mg; prewashed with methanol) in methanol(800 μL) was added. The mixture was stirred at room temperature underargon for 17 h, diluted with ethyl acetate/ether (1:1, 4 mL) anddecanted. The resin was washed with ether (8 mL) and the combinedorganic phases washed with brine and saturated NaHCO₃, dried (MgSO₄) andevaporated. The residue was purified by HPLC (6.2 mm×25 cm Zorbax-Silcolumn, 4 mL/min) using hexane/2-propanol (9:1) solvent system.Analytically pure 2-methylene-19-nor-vitamin 11 (2.3 mg, 97%) wascollected at R_(V) 29 mL (1α,25-dihydroxyvitamin D₃ was eluted at R_(v)52 mL in the same system) as a white solid: UV (in EtOH) λ_(max) 243.5,252, 262.5 nm; ¹H NMR (CDCl₃) δ 0.552 (3H, s, 18-H₃), 0.941 (3H, d,J=6.4 Hz, 21-H₃), 1.222 (6H, s, 26- and 27-H₃), 2.01 (2H, m), 2.27-2.36(2H, m), 2.58 (1H, m), 2.80-2.88 (2H, m), 4.49 (2H, m, 1β- and 3α-H),5.10 and 5.11 (1H and 1H, each s, ═CH₂), 5.89 and 6.37 (1H and 1H, eachd, J=11.3 Hz, 7- and 6-H); MS m/z (relative intensity) 416 (M⁺, 83), 398(25), 384 (31), 380 (14), 351 (20), 313 (100); exact mass calcd forC₂₇H₄₄O₃ 416.3290, found 416.3279.

[0097] (i) Hydrogenation of 2-methylene-19-nor-vitamin 11.

[0098] 1α,25-Dihydroxy-2α- and 1α,25-Dihydroxy-2β-methyl-19-nor-vitaminD₃ (12 and 13). Tris(triphenylphosphine)rhodium(I) chloride (2.3 mg, 2.5μmol) was added to dry benzene (2.5 ml) presaturated with hydrogen. Themixture was stirred at room temperature until a homogeneous solution wasformed (ca. 45 min). A solution of vitamin 11 (1.0 mg, 2.4 μmol) in drybenzene (0.5 mL) was then added and the reaction was allowed to proceedunder a continuous stream of hydrogen for 3 h. Benzene was removed undervacuum, and hexane/ethyl acetate (1:1, 2 mL) was added to the residue.The mixture was applied on a silica Sep-Pak and both 2-methyl vitaminswere eluted with the same solvent system (20 mL). Further purificationwas achieved by HPLC (6.2 mm×25 cm Zorbax-Sil column, 4 mL/min) usinghexane/2-propanol (9:1) as a solvent system. The mixture (ca. 1:1) of2-methyl-19-nor-vitamins (2α- and 2β-epimers 12 and 13; 0.80 mg, 80%)gave a single peak at R_(v) 33 mL.

[0099] 12 and 13: UV (in EtOH) λ_(max) 243, 251, 261.5 nm; ¹H NMR(CDCl₃) δ 0.536 and 0.548 (3H and 3H, each s, 2×18-H₃), 0.937 (6H, d,J=6.3 Hz, 2×21-H₃), 1.133 and 1.144 (3H and 3H, each d, J˜6 Hz,2×2-CH₃), 1.219 [12H, s, 2× (26- and 27-H₃)], 2.60 (1H, dd, J=13.0, 4.6Hz), 2.80 (3H, m), 3.08 (1H, dd, J=12.6, 4.0 Hz), 3.51 (1H, dt, J=4.6,10.2 Hz), 3.61 (1H, dt, J=4.5, 9.1 Hz), 3.90 (1H, narr m), 3.96 (1H,narr m), 5.82, 5.87, 6.26, and 6.37 (each 1H, each d, J=11.2 Hz); MS m/z(relative intensity) 418 (M, 100), 400 (25), 385 (15), 289 (30), 245(25).

[0100] Separation of both epimers was achieved by reversed-phase HPLC(10 mm×25 cm Zorbax-ODS column, 4 mL/min) using methanol/water (85:15)solvent system. 2β-Methyl vitamin 13 (0.35 mg, 35%) was collected atR_(v) 41 mL and its 2α-epimer 12 (0.34 mg, 34%) at R_(v) 46 mL.

[0101] 12: UV (in EtOH) λ_(max) 243, 251, 261 nm; ¹H NMR (CDCl₃) δ 0.536(3H, s, 18-H₃), 0.937 (3H, d, J=6.4 Hz, 21-H₃), 1.134 (3H, d, J=6.9 Hz,2α-CH₃), 1.218 (6H, s, 26- and 27-H₃), 2.13 (1H, -t, J˜12 Hz, 40-H),2.22 (1H, br d, J=13 Hz, 10β-H), 2.60 (1H, dd, J=12.8,4.3 Hz, 4α-H),2.80 (2H, m, 9β- and 10α-H), 3.61 (1H, m, w/2=24 Hz, 3α-H), 3.96 (1H, m,w/2=12 Hz, 1β-H), 5.82 and 6.37 (1H and 1H, each d, J=11.1 Hz, 7- and6-H); MS m/z (relative intensity) 418 (M⁺, 62), 400 (32), 385 (17), 289(36), 271 (17), 253 (20), 245 (43), 69 (100), 59 (74) ); exact masscalcd for C₂₇H₄₆O₃ 418.3447, found 418.3441.

[0102] 13: UV (in EtOH) λ_(max) 242, 250.5, 261 nm; 1H NMR (CDCl₃) δ0.548 (3H, s, 18-H₃), 0.940 (3H, d, J=6.4 Hz, 21-H₃), 1.143 (3H, d,J=6.8 Hz, 2,-CH₃), 1.220 (6H, s, 26- and 27-H₃), 2.34 (1H, dd, J=13.7,3.3 Hz, 4β-H), 2.43 (1H, br d, J=13.7 Hz, 4α-H), 2.80 (1H, dd, J=12 and4 Hz, 9-H), 3.08 (1H, dd, J=13.0, 4.2 Hz, 10β-H), 3.51 (1H, m, w/2=25Hz, 1β-H), 3.90 (1H, m, w/2=11 Hz, 3α-H), 5.87 and 6.26 (1H and 1H, eachd, J=11.2 Hz, 7- and 6-H); MS m/z (relative intensity) 418 (M⁺, 63), 400(47), 385 (16), 289 (40), 271 (32), 253 (27), 245 (47), 69 (100), 59(64); exact mass calcd for C₂₇H₄₆O₃ 418.3447, found 418.3436.

EXAMPLE 2

[0103] Preparation of 20(S)-1α,25-dihydroxy-2α- and20(S)-1α,25-dihydroxy-2,-methyl-19-nor-vitamin D₃ (18 and 19).

[0104] SCHEME II illustrates the preparation of protected20(S)-25-hydroxy Grundmann's ketone 15, its coupling with phosphineoxide 8 (obtained as described in Example 1) and selective hydrogenationof exomethylene group in 2-methylene compound 17.

[0105] (a) Silylation of Hydroxy Ketone 14.

[0106] 20(S)-25-[(Triethylsilyl)oxy]-des-A,B-cholestan-8-one (15). Asolution of the ketone 14 (Tetrionics, Inc.; 56 mg, 0.2 mmol) andimidazole (65 mg, 0.95 mmol) in anhydrous DMF (1.2 μL) was treated withtriethylsilyl chloride (95 μL, 0.56 mmol), and the mixture was stirredat room temperature under argon for 4 h. Ethyl acetate was added andwater, and the organic layer was separated. The ethyl acetate layer waswashed with water and brine, dried (MgSO₄) and evaporated. The residuewas passed through a silica Sep-Pak cartridge in hexane/ethyl acetate(9:1), and after evaporation, purified by HPLC (9.4 mm×25 cm Zorbax-Silcolumn, 4 mL/min) using hexane/ethyl acetate (9:1) solvent system. Pureprotected hydroxy ketone 15 (55 mg, 70%) was eluted at R_(v) 35 mL as acolorless oil: ¹H NMR (CDCl₃) δ 0.566 (6H, q, J=7.9 Hz, 3× SiCH₂), 0.638(3H, s, 18-H₃), 0.859 (3H, d, J=6.0 Hz, 21-H₃), 0.947 (9H, t, J=7.9 Hz,3× SiCH₂CH₃), 1.196 (6H, s,26- and 27-H₃), 2.45 (1H, dd, J=11.4, 7.5 Hz,14α-H).

[0107] (b) Wittig-Horner Coupling of Protected 20(S)-25-hydroxyGrundmann's Ketone With the Phosphine Oxide 8.

[0108] 20(S)-1α,25-Dihydroxy-2-methylene-19-nor-vitamin D₃ (17). To asolution of phosphine oxide 8 (15.8 mg, 27.1 μmol) in anhydrous THF (200μL) at 0° C. was slowly added n-BuLi (2.5 M in hexanes, 11 μL, 27.5μmol) under argon with stirring. The solution turned deep orange. Themixture was cooled to −78° C. and a precooled (−78° C.) solution ofprotected hydroxy ketone 15 (8.0 mg, 20.3 μmol) in anhydrous THF (100μL) was slowly added. The mixture was stirred under argon at −78° C. for1 h and at 0° C. for 18 h. Ethyl acetate was added, and the organicphase was washed with brine, dried (MgSO₄) and evaporated. The residuewas dissolved in hexane and applied on a silica Sep-Pak cartridge, andwashed with hexane/ethyl acetate (99.5:0.5,20 mL) to give 19-nor-vitaminderivative 16 (7 mg, 45%) as a colorless oil. The Sep-Pak was thenwashed with hexane/ethyl acetate (96:4, 10 mL) to recover some unchangedC,D-ring ketone 15 (4 mg), and with ethyl acetate (10 mL) to recoverdiphenylphosphine oxide (9 mg). For analytical purpose a sample ofprotected vitamin 16 was further purified by BPLC (6.2 mm×25 cmZorbax-Sil column, 4 mL/min) using hexane/ethyl acetate (99.9:0.1)solvent system.

[0109] 16: UV (in hexane) λ_(max) 244, 253.5, 263 nm; ¹H NMR (CDCl₃) δ0.026, 0.049, 0.066, and 0.080 (each 3H, each s, 4× SiCH₃), 0.541 (3H,s, 18-H₃), 0.564 (6H, q, J=7.9 Hz, 3× SiCH₂), 0.848 (3H, d, J=6.5 Hz,21-H₃), 0.864 and 0.896 (9H and 9H, each s, 2× Si-t-Bu), 0.945 (9H, t,J=7.9 Hz, 3× SiCH₂CH₃), 1.188 (6H, s, 26- and 27-H₃), 2.15-2.35 (4H, brm), 2.43-2.53 (3H, br m), 2.82 (1H, br d, J=12.9 Hz, 9β-H), 4.42 (2H, m,1β- and 3α-H), 4.92 and 4.97 (11H and 11H, each s, ═CH₂), 5.84 and 6.22(1H and 1H, each d, 3=11.1 Hz, 7- and 6-H); MS m/z (relative intensity)758 (M⁺, 33), 729 (M⁺-Et, 7), 701 (M⁺-t-Bu, 5), 626 (100), 494 (25), 366(52), 75 (82), 73 (69).

[0110] Protected vitamin 16 (5.0 mg) was dissolved in benzene (160 μL)and the resin (AG 50W-X4, 70 mg; prewashed with methanol) in methanol(900 μL) was added. The mixture was stirred at room temperature underargon for 19 h, diluted with ethyl acetate/ether (1:1, 4 mL) anddecanted. The resin was washed with ether (8 mL) and the combinedorganic phases washed with brine and saturated NaHCO₃, dried (MgSO₄) andevaporated. The residue was purified by HPLC (6.2 mm×25 cm Zorbax-Silcolumn, 4 mL/min) using hexane/2-propanol (9:1) solvent system.Analytically pure 2-methylene-19-nor-vitamin 17 (2.6 mg, 95%) wascollected at R_(v) 28 mL [(20R)-analog was eluted at R_(v) 29 mL and1α,25-dihydroxyvitamin D₃ at R_(v) 52 mL in the same system] as a whitesolid: WV (in EtOH) λ_(max) 243.5, 252.5, 262.5 nm; 1H NMR (CDCl₃) δ0.551 (3H, s, 18-H₃), 0.858 (3H, d, J=6.6 Hz, 21-H₃), 1.215 (6H, s, 26-and 27-H₃), 1.95-2.04 (2H, m), 2.27-2.35 (2H, m), 2.58 (1H, dd, J=13.3,3.7 Hz), 2.80-2.87 (2H, m), 4.49 (2H, m, 1β- and 3α-H), 5.09 and 5.11(1H and 1H, each s, ═CH₂), 5.89 and 6.36 (1H and 1H, each d, J=11.3 Hz,7- and 6-11); MS m/z (relative intensity) 416 (M⁺, 100), 398 (26), 380(13), 366 (21), 313 (31); exact mass calcd for C₂₇H₄₄O₃ 416.3290, found416.3275.

[0111] (c) Hydrogenation of 2-methylene-19-nor-vitamin 17.

[0112] 20(S)-1α,25-Dihydroxy-2α- and20(S)-1α,25-Dihydroxy-2β-methyl-19-nor-vitamin D₃ (18 and 19).Tris(triphenylphosphine)rhodium(l) chloride (2.3 mg, 2.5 μmol) was addedto dry benzene (2.5 mL) presaturated with hydrogen. The mixture wasstirred at room temperature until a homogeneous solution was formed (ca.45 min). A solution of vitamin 17 (1.0 mg, 2.4 μmol) in dry benzene (0.5mL) was then added and the reaction was allowed to proceed under acontinuous stream of hydrogen for 3 h. Benzene was removed under vacuum,and hexane/ethyl acetate (1:1, 2 mL) was added to the residue. Themixture was applied on a silica Sep-Pak and both 2-methyl vitamins wereeluted with the same solvent system (20 mL). Further purification wasachieved by HPLC (6.2 mm×25 cm Zorbax-Sil column, 4 mL/min) usinghexane/2-propanol (9:1) as a solvent system. The mixture (ca. 1:1) of2-methyl-19-nor-vitamins (2α- and 2β-epimers 18 and 19; 0.43 mg, 43%)gave a single peak at R_(v) 31 mL.

[0113] 18 and 19: WV (in EtOH) λ_(max) 243, 251, 261 nm; ¹H NMR (CDCl₃)δ 0.534 and 0.546 (3H and 3H, each s, 2×18-H₃), 0.852 and 0.857 (3H and3H, each d, J=6.5 Hz, 2×21-H₃), 1.133 (3H, d, J=6.7 Hz, 2-CH₃), 1.143(3H, d, J=6.5 Hz, 2-CH₃), 1.214 [12H, s, 2× (26- and 27-H₃)], 2.60 (1H,dd, J=12.7, 4.5 Hz), 2.80 (3H, m), 3.08 (1H, dd, J=13.1, 4.3 Hz), 3.51(1H, br m; after D₂0 dt, J=4.5, 10.0 Hz), 3.61 (1H, br m; after D₂O dt,J=4.4, 9.2 Hz), 3.90 (1H, narr m), 3.96 (1H, narr m), 5.82, 5.87, 6.26,and 6.37 (each 1H, each d, J=11.3 Hz); MS in/z (relative intensity) 418(M⁺, 100), 400 (45), 385 (20), 289 (38), 245 (47).

[0114] Separation of both epimers was achieved by reversed-phase HPLC(10 mm×25 cm Zorbax-ODS column, 4 mL/min) using methanol/water (85:15)solvent system. 2β-Methyl vitamin 19 (16%) was collected at R_(V) 36 mLand its 2α-epimer 18 (20%) at R_(V) 45 mL.

[0115] 18: WV (in EtOH) λ_(max) 242.5, 251, 261 nm; ¹H NMR (CDCl₃) δ0.534 (3H, S, 18-H₃), 0.852 (3H, d, J=6.6 Hz, 21-H₃), 1.133 (3H, d,J=6.9 Hz, 2α-CH₃), 1.214 (6H, s, 26- and 27-H₃), 2.13 (1H, ˜t, J˜12 Hz,4β-1H), 2.22 (1H, br d, J=13 Hz, 10β-H), 2.60 (1H, dd, J=12.8, 4.4 Hz,4β-H), 2.80 (2H, m, 9β- and 10α-H), 3.61 (1H, m, w/2=25 Hz, 3α-H), 3.95(1H, m, w/2=11 Hz, 1β-H), 5.82 and 6.37 (1H and 1H, each d, J=11.2 Hz,7- and 6-H); MS m/z (relative intensity) 418 (M⁺, 58), 400 (25), 385(20), 289 (28), 271 (23), 253 (22), 245 (38), 69 (100), 59 (47) ); exactmass calcd for C₂₇H₄₆O₃ 418.3447, found 418.3450.

[0116] 19: UV (in EtOH) λ_(max) 242.5, 250.5, 261 nm; ¹H NMR (CDCl₃) δ0.547 (3H, s, 18-H₃), 0.857 (3H, d, J=6.6 Hz, 21-H₃), 1.143 (3H, d,J=6.8 Hz, 2β-CH₃), 1.214 (6H, s, 26- and 27-H₃), 2.34 (1H, dd, J=13.8,3.1 Hz, 413-H), 2.43 (1H,.br d, J=13.8 Hz, 4α-H), 2.80 (1H, dd, J=12 and4 Hz, 9β-H), 3.08 (1H, dd, J=12.9, 4.4 Hz, 10β-H), 3.50 (1H, m, w/2=26Hz, 1,-H), 3.89 (1H, m, w/2=11 Hz, 3α-H), 5.86 and 6.26 (1H and 1H, eachd, J=11.2 Hz, 7- and 6-H); MS m/z (relative intensity) 418 (M⁺, 68), 400(47), 385 (21),289 (33), 271 (27), 253 (26), 245 (47), 69 (100), 59(53); exact mass calcd for C₂₇H₄₆O₃ 418.3447, found 418.3448.

[0117] 1α,25-Dihydroxy-2α- and1α,25-Dihydroxy-2β-(hydroxymethyl)-19-norvitamin D₃ (20 and 21).9-Borabicyclo[3.3.1]nonane (0.5 M in THF, 60 μL, 30 μmol) was added to asolution of vitamin 11 (1.25 mg, 3 μmol) in anhydrous THF (50 μL) atroom temperature (evolution of hydrogen was observed). After 3 h ofstirring, the mixture was quenched with methanol (20 μL), stirred for 15min at room temperature, cooled to 0° C., and treated successively with6 M NaOH (10 μL, 60 μmol) and 30% H₂O₂ (10 μL). The mixture was heatedfor 1 h at 55° C., cooled, benzene and brine were added, and the organicphase was separated, dried and evaporated. The crystalline residue wasdissolved in ether (0.5 mL) and kept in freezer overnight. The ethersolution was carefully removed from the precipitated crystals ofcyclooctanediol and evaporated. Separation of the residue was achievedby HPLC (6.2 mm×25 cm Zorbax-Sil column, 4 mL/min) usinghexane/2-propanol (85:15) solvent system. Traces of unreacted substrate11 were eluted at R_(V) 16 mL, whereas isomeric 2-hydroxymethyl vitamins20 and 21 were collected at R_(V) 33 mL and 40 mL, respectively. Furtherpurification of both products by reversed-phase HPLC (10 mm×25 cmZorbax-ODS column, 4 mL/min) using methanol/water (9:1) solvent systemafforded analytically pure vitamin 20 (0.14 mg, 11%) and its 2β-isomer21 (0.31 mg, 24%) collected at R_(V) 26 mL and 23 mL, respectively.

[0118] 20: UV (in EtOH) λ_(max) 242.5, 250.5, 261 nm; ¹H NMR (CDCl₃) δ0.536 (3H, s, 18-H₃), 0.939 (3H, d, J=6.4 Hz, 21-H₃), 1.214 (6H, s, 26-and 27-H₃), 2.13 (1H, br d, J=13.5 Hz, 10-H), 2.21 (1H, ˜t, J=12 Hz,4β-H), 2.64 (1H, dd, J=12.7, 4.5 Hz, 4α-H), 2.80 (2H, br d, J=12.7 Hz,9β-H), 2.90 (1H, br d, J=13.5 Hz, 10α-H), 3.95-4.1 (3H, br m, 2α-CH₂OHand 3α-H), 4.23 (1H, m, w/2=11 Hz, 1β-H), 5.79 and 6.41 (1H and 1H, eachd, J=11.1 Hz, 7- and 6-H); MS m/z (relative intensity) 434 (M⁺, 37), 416(33), 398 (16), 383 (10), 305 (12), 287 (25), 269 (26), 245 (40), 69(100), 59 (74); exact mass calcd for C₂₇H₄₆O₄ 434.3396, found 434.3397.

[0119] 21: UV (in EtOH) λ_(max) 242, 250.5, 260.5 nm; ¹H NMR (CDCl₃) δ0.553 (3H, s, 18-H₃), 0.942 (3H, d, J=6.5 Hz, 21-H₃), 1.220 (6H, s, 26-and 27-H₃), 2.31 (1H, br d, J=14 Hz, 4β-H), 2.45 (1H, br d, J=14 Hz,4α-H), 2.79 (1H, br d, J=13 Hz, 9-H), 3.17 (1H, dd, J=12.8,4.2 Hz,10β-H), 3.95-4.1 (3H, br m, 1β-H and 2β-CH₂OH), 4.17 (1H, m, w/2=10 Hz,3α-H), 5.89 and 6.26 (1H and 1H, each d, J=11.0 Hz, 7- and 6-H); MS m/z(relative intensity) 434 (M⁺, 50), 416 (46), 398 (20), 383 (11), 305(14), 287 (26), 269 (30), 245 (50), 69 (100), 59 (75); exact mass calcdfor C₂₇H₄₆O₄ 434.3396, found 434.3402.

[0120] 20(S)-1α,25-Dihydroxy-2α- and20(S)-1α,25-Dihydroxy-2β-(hydroxymethyl)-19-norvitamin D₃ (22 and 23).The hydroboration of 20(S)-vitamin 17 and subsequent oxidation of theorganoborane adduct were performed using the procedure analogous to thatdescribed above for (20R)-epimer 11. The reaction products wereseparated by HPLC (6.2 mm×25 cm Zorbax-Sil column, 4 mL/min) usinghexane/2-propanol (87.5:12.5) solvent system, and the isomeric2-hydroxymethyl vitamins 22 and 23 were collected at R_(V) 40 mL and 47mL, respectively. Further purification of both products byreversed-phase HPLC (10 mm×25 cm Zorbax-ODS column, 4 mL/min) usingmethanol/water (9:1) solvent system afforded analytically pure vitamin22 (9%) and its 2β-isomer 23 (26%) collected at R_(V) 25 mL and 22 mL,respectively.

[0121] 22: UV (in EtOH) λ_(max) 242.5, 250.5, 261 nm; ¹H NMR (CDCl₃) δ0.532 (3H, s, 18-H₃), 0.853 (3H, d, J=6.6 Hz, 21-H₃), 1.214 (6H, s, 26-and 27-H₃), 2.13 (1H, br d, J=13.3 Hz, 10β-H), 2.21 (1H, t, J=12 Hz,4β-H), 2.64 (1H, dd, J=12.8, 4.3 Hz, 4α-H), 2.80 (1H, br d, J=12 Hz,913-H), 2.90 (1H, br d, J=13.3 Hz, 10α-H), 3.95-4.1 (3H, br m, 2α-CH₂OHand 3α-H), 4.24 (1H, m, w/2=10 Hz, 1β-H), 5.81 and 6.43 (1H and 1H, eachd, J=11.1 Hz, 7- and 6-H); MS m/z (relative intensity) 434 (M⁺, 41), 416(34), 398 (16), 383 (10), 305 (10), 287 (28), 269 (26), 245 (51), 69(100), 59 (82); exact mass calcd for C₂₇H₄₆O₄ 434.3396, found 434.3390.

[0122] 23: UV (in EtOH) λ_(max) 242, 250.5, 260.5 nm; ¹H NMR (CDCl₃) δ0.551 (3H, s, 18-H₃), 0.861 (3H, d, J=6.4 Hz, 21-H₃), 1.215 (6H, s, 26-and 27-H₃), 2.31 (1H, br d, J=13.7 Hz, 413-H), 2.45 (1H, br d, J=13.7Hz, 4α-H), 2.80 (1H, br d, J=12.5 Hz, 9β-H, 3.17 (1H, dd, J=12.7,4.2 Hz,10β-H), 3.95-4.1 (3H, br m, 1β-H and 2β-CH₂OH), 4.17 (1H, m, w/2=10 Hz,3α-H), 5.89 and 6.26 (1H and 1H, each d, J=11.1 Hz, 7- and 6-H); MS m/z(relative intensity) 434 (, 35), 416 (29), 398 (15), 383 (8), 305 (10),287 (18), 269 (23), 245 (48), 69 (100), 59 (93); exact mass calcd forC₂₇H₄₆O₄ 434.3396, found 434.3408.

Biological Activity of 2-methyl-Substituted 19-nor-1,25-(OH)₂D₃Compounds and Their 2OS-Isomers

[0123] The synthesized 2-substituted vitamins were tested for theirability to bind the porcine intestinal vitamin D receptor (See FIGS. 1,3 and 5). A comparison between the natural hormone 1α,25-(OH)₂D₃ and2-methyl substituted 19-norvitamins 12, 18 and 19 shows that they areabout as active as 1α,25-(OH)₂D₃, while the 2β-methyl isomer in the20R-series 13 is 39-fold less effective. The 2α-hydroxymethyl vitamin Danalog 22 with the “unnatural” configuration at C-20 was almostequivalent to 1α,25-(OH)₂D₃ with respect to receptor binding, and theisomeric 23 proved to be less potent (6-8×) than these compounds. Thecorresponding 2α-hydroxymethyl analog possessing the “natural”20R-configuration 20 exhibited about the same binding affinity as 23,whereas the 2β-isomer 21 was ca. 8 times less effective. The foregoingresults of the competitive binding analysis show that vitamins with theaxial orientation of the 1α-hydroxy group exhibit a significantlyenhanced affinity for the receptor.

[0124] It might be expected from these results that all of thesecompounds would have equivalent biological activity. Surprisingly,however, the 2-methyl substitutions produced highly selective analogswith their primary action on bone. When given for 7 days in a chronicmode, the most potent compounds tested were a mixture of the α and βisomers of 2-methyl 19-nor-20S-1,25-(OH)₂D₃ (Table 1). When given at 130μmol/day, the activity of this mixture of compounds on bone calciummobilization (serum calcium) was much higher than that of the nativehormone, possibly as high as 10 or 100 times higher. Under identicalconditions, twice the dose of 1,25-(OH)₂D₃ gave a serum calcium value of7.2 mg/100 ml, while a mixture of 2-methyl-(α andβ)-19-nor-20S-1,25-(OH)₂D₃ gave a value of 9.6 mg/100 ml of serumcalcium at the 130 μmol dose. When given at 260 μmol/day, this mixtureproduced the astounding value of 12.2 mg/100 ml of serum calcium at theexpense of bone. To show its selectivity, these compounds produced nosignificant change in intestinal calcium transport at 130 μmol doselevel while having a strong bone calcium mobilizing activity. At thehigher dose, the 2-methyl-20S mixture did produce an intestinaltransport response but gave an enormous bone mobilization response. Amixture of the α and β isomers of 2-methyl-19-nor-1,25-(OH)₂D₃ also hadstrong bone calcium mobilization at both dose levels but also showed nointestinal calcium transport activity. Thus, the 2-methyl-α and βderivatives given as a mixture showed strong preferential bone calciummobilizing activity especially when the side chain was in the20S-configuration. These results illustrate that the 2-methyl and the20S-2-methyl derivatives of 19-nor-1,25-(OH)₂D₃ are selective for themobilization of calcium from bone. Table 2 illustrates the response ofboth intestine and serum calcium to a single large dose of the variouscompounds; again, supporting the conclusions derived from Table 1.

[0125] The results in FIG. 2 illustrate that a mixture of the α and βderivatives of 2-methyl-19-nor-20S-1,25-(OH)₂D₃ is extremely potent ininducing differentiation of HL-60 cells to the moncyte. The 2-methyl-αand β compounds had activity similar to 1,25-(OH)₂D₃. These resultsillustrate the potential of the 2-methyl-19-nor-20S-1,25-(OH)₂D₃compounds as anti-cancer agents, especially against leukemia, coloncancer, breast cancer and prostate cancer, or as agents in the treatmentof psoriasis.

[0126] Competitive binding of the analogs to the porcine intestinalreceptor was carried out by the method described by Dame et al(Biochemnistry 25, 4523-4534, 1986).

[0127] The differentiation of HL-60 promyelocytic into monocytes wasdetermined as described by Ostrem et al (J. Biol. Chem. 262,14164-14171, 1987). TABLE 1 Response of Intestinal Calcium Transport andSerum Calcium (Bone Calcium Mobilization) Activity to Chronic Doses of2-Methyl Derivatives of 19-Nor-1,25-(OH)₂D₃ and its 20S IsomersIntestinal Calcium Dose Transport Serum Calcium Group (pmol/day/7 days)(S/M) (mg/100 ml) Vitamin D Deficient Vehicle 5.5 ± 0.2  5.1 ± 0.161,25-(OH)₂D₃ Treated 260 6.2 ± 0.4 7.2 ± 0.5 2-Methyl (α and β) 130 5.0± 0.3 6.1 ± 0.1 19-Nor-1,25-(OH)₂D₃ 260 5.3 ± 0.6 6.7 ± 0.4 2-Methyl (αand β) 130 5.0 ± 0.9 9.6 ± 0.1 19-Nor-20S-1,25-(OH)₂ 260 6.9 ± 0.5 12.2± 0.3  D₃

[0128] Male weanling rats were obtained from Sprague Dawley Co.(Indianapolis, Ind.) and fed a 0.47% calcium, 0.3% phosphorus vitaminD-deficient diet for 1 week and then given the same diet containing0.02% calcium, 0.3% phosphorus for 2 weeks. During the last week theywere given the indicated dose of compound by intraperitoneal injectionin 0.1 ml 95% propylene glycol and 5% ethanol each day for 7 days. Thecontrol animals received only the 0.11 ml of 95% propylene glycol, 5%ethanol. Twenty-four hours after the last dose, the rats were sacrificedand intestinal calcium transport was determined by everted sac techniqueas previously described and serum calcium determined by atomicabsorption spectrometry on a model 3110 Perkin Elmer instrument Norwalk,Conn.). There were 5 rats per group and the values represent mean ±SEM.TABLE 2 Response of Intestinal Calcium Transport and Serum Calcium (BoneCalcium Mobilization) Activity to a Single Dose of the2-Methyl-Derivatives of 19-Nor-1,25-(OH)₂D₃ and its 20S IsomersIntestinal Calcium Transport Serum Calcium Group (S/M) (mg/100 ml) -DControl 4.2 ± 0.3 4.7 ± 0.1 1,25-(OH)₂D₃ 5.8 ± 0.3 5.7 ± 0.2 2-Methyl (αand β mixture)-19-Nor- 3.6 ± 0.4 5.4 ± 0.1 1,25-(OH)₂D₃ 2-Methyl (α andβ mixture)-19-Nor- 6.7 ± 0.6 8.1 ± 0.3 20S-1,25-(OH)₂D₃

[0129] Male Holtzman strain weanling rats were obtained from the SpragueDawley Co. (Indianapolis, Ind.) and fed the 0.47% calcium, 0.3%phosphorus diet described by Suda et al. (J. Nutr. 100 1049-1052, 1970)for 1 week and then fed the same diet containing 0.02% calcium and 0.3%phosphorus for 2 additional weeks. At this point, they received a singleintrajugular injection of the indicated dose dissolved in 0.1 ml of 95%propylene glycol/5% ethanol. Twenty-four hours later they weresacrificed and intestinal calcium transport and serum calcium weredetermined as described in Table 1. The dose of the compounds was 650μmol and there were 5 animals per group. The data are expressed as mean±SEM.

[0130] When given for 7 days in a chronic mode, the most potentindividual compound tested was 2α-methyl 19-nor-20S-1,25-(OH)₂D₃ (Table3). When given at 130 μmol/day, the activity of this compound on bonecalcium mobilization (serum calcium) was much higher than that of thenative hormone, possibly as high as 10 or 100 times higher. Underidentical conditions, twice the dose of 1,25-(OH)₂D₃ gave a serumcalcium value of 6.6±0.4 mg/100 ml, while2α-methyl-19-nor-20S-1,25-(OH)₂D₃ gave a value of 8.3±0.7 mg/100 ml ofserum calcium at the 130 μmol dose. When given at 260 μmol/day,2α-methyl-19-nor-20S-1,25-(OH)₂D₃ produced the astounding value of10.3±0.11 mg/100 ml of serum calcium at the expense of bone. To show itsselectivity, this compound also produced a significant change inintestinal calcium transport at both the 260 μmol and the 130 μmol doselevels while having a strong bone calcium mobilizing activity. At thehigher dose, the 2α-methyl-20S compound did produce a significantintestinal transport response but also gave an enormous bonemobilization response. With respect to the 2β-methyl-19-nor-20Scompound, the data in Table 3 show it has little, if any, intestinalcalcium transport activity, and little, if-any, bone mobilizationactivity. The data in Table 4 illustrate that2α-methyl-19-nor-1,25-(OH)₂D₃ also had relatively strong bone calciummobilization at both dose levels and also showed some intestinal calciumtransport activity. In contrast, 2β-methyl-19-nor-1,25-(OH)₂D₃ showedlittle, if any, intestinal calcium transport or bone calciummobilization activities. Thus, the 2α-methyl-19-nor derivative showedstrong preferential bone calcium mobilizing activity especially when theside chain was in the 208-configuration. These results illustrate thatthe 2α-methyl and the 20S-2α-methyl derivatives of 19-nor-1,25-(OH)₂D₃are selective for the mobilization of calcium from bone.

[0131] The results in FIG. 4 illustrate that2α-methyl-19-nor-20S-1,25-(OH)₂D₃ and 2α-methyl-19-nor-1,25-(OH)₂D₃ areextremely potent in inducing differentiation of HL-60 cells to themonocyte. The 2β-methyl compounds had activity similar to 1,25-(OH)₂D₃.These results illustrate the potential of the2α-methyl-19-nor-20S-1,25-(OH)₂D₃ compound as an anti-cancer agent,especially against leukemia, colon cancer, breast cancer and prostatecancer, or as an agent in the treatment of psoriasis.

[0132] Competitive binding of the analogs to the porcine intestinalreceptor was carried out by the method described by Dame et al(Biochemistry 25, 4523-4534, 1986).

[0133] The differentiation of HL-60 promyelocytic into monocytes wasdetermined as described by Ostrem et al (J. Biol. Chem. 262,14164-14171, 1987). TABLE 3 Response of Intestinal Calcium Transport andSerum Calcium (Bone Calcium Mobilization) Activity to Chronic Doses ofthe 20S Isomers of 2-Methyl Derivatives of 19-Nor-1,25-(OH)₂D₃Intestinal Dose Calcium (pmol/ Transport Serum Calcium Group day/7 days)(S/M) (mg/100 ml) Vitamin D Deficient Vehicle 2.9 ± 0.2 4.2 ± 0.11,25-(OH)₂D₃ Treated 260 4.6 ± 0.2 6.6 ± 0.4 2α-Methyl-19-nor-20(S)- 13012.9 ± 1.9  8.3 ± 0.7 1,25-(OH)₂D₃ 260 8.4 ± 1.1 10.3 ± 0.112β-Methyl-19-nor-20(S)- 130 2.9 ± 0.3 4.4 ± 0.1 1,25-(OH)₂D₃ 260 3.8 ±0.1 4.4 ± 0.1

[0134] With respect to the data in Tables 3 and 4, male weanling ratswere obtained from Sprague Dawley Co. (Indianapolis, Ind.) and fed a0.47% calcium, 0.3% phosphorus vitamin D-deficient diet for 1 week andthen given the same diet containing 0.02% calcium, 0.3% phosphorus for 2weeks. During the last week they were given the indicated dose ofcompound by intraperitoneal injection in 0.1 ml 95% propylene glycol and5% ethanol each day for 7 days. The control animals received only the0.1 ml of 95% propylene glycol, 5% ethanol. Twenty-four hours after thelast dose, the rats were sacrificed and intestinal calcium transport wasdetermined by everted sac technique as previously described and serumcalcium determined by atomic absorption spectrometry on a model 3110Perkin Elmer instrument (Norwalk, Conn.). There were 5 rats per groupand the values represent mean ±SEM. TABLE 4 Response of IntestinalCalcium Transport and Serum Calcium (Bone Calcium Mobilization) Activityto Chronic Doses of the 2-Methyl Derivatives of 19-Nor-1,25-(OH)₂D₃Intestinal Calcium Dose Transport Serum Calcium Group Pmol/day/7 days(S/M) (mg/100 ml) -D Control  0 2.3 ± 0.8 3.9 ± 0.2 1,25-(OH)₂D₃ 260 5.6± 1.3 6.1 ± 0.5 2α-Methyl-19-nor- 130 4.3 ± 1.0 4.8 ± 0.2 1,25-(OH)₂D₃260 5.3 ± 1.3 5.8 ± 0.5 2β-Methyl-19-nor- 130 4.4 ± 0.8 4.1 ± 0.11,25-(OH)₂D₃ 260 3.1 ± 0.9 3.8 ± 0.2

[0135] Table 5 provides intestinal calcium transport and bone calciummobilization data for 2-hydroxymethyl derivatives of19-nor-1α,25-(OH)₂D₃. These derivatives turned out to be relativelyinactive, including those in the 20S-series 22 and 23. TABLE 5 Responseof Intestinal Calcium Transport and Serum Calcium (Bone CalciumMobilization) Activity to Chronic Doses of the2-Hydroxymethyl-Derivatives of 19-Nor-1,25-(OH)₂D₃ Intestinal CalciumDose Transport Serum Calcium Group Pmol/day/7 days (S/M) (mg/100 ml)Vitamin D Deficient Vehicle 4.0 ± 0.3 3.8 ± 0.1 1,25-(OH)₂D₃ Treated 2606.6 ± 0.5 5.2 ± 0.1 2α-Hydroxymethyl-19- 130 5.0 ± 0.3 4.0 ± 0.1nor-20(S)-1,25-(OH)₂D₃ 260 5.8 ± 0.4 3.9 ± 0.1 2β-Hydroxymethyl-19- 1303.5 ± 0.7 3.6 ± 0.1 nor-20(S)-1,25-(OH)₂D₃ 260 3.5 ± 0.3 3.5 ± 0.2

[0136] In the next assay, the cellular activity of the synthesizedcompounds was established by studying their ability to inducedifferentiation of human promyelocyte HL-60 cells into monocytes. It wasfound that all of the synthesized vitamin D analogs with the “unnatural”20S-configuration were more potent than 1α,25-(OH)₂D₃. Moreover, thesame relationship between cellular activity and conformation of thevitamin D compounds was established as in the case of receptor bindinganalysis and in vivo studies, i.e. 2α-substituted vitamin D analogs wereconsiderably more active than their 2β-substituted counterparts with theequatorially oriented 1α-hydroxy group. Thus, 2α-methyl vitamins 12 and18 proved to be 100 and 10 times, respectively, more active than theircorresponding 2β-isomers 13 and 19 in the cultures of HL-60 in vitro,whereas in the case of 2-hydroxymethyl derivatives (20, 22 versus 21,23) these differences were slightly smaller. Since vitamins with2β-methyl substituent (13, 19) and both 2-hydroxymethyl analogs in20S-series (22, 23) have selective activity profiles combining highpotency in cellular differentiation, and lack of calcemic activity, suchcompounds are potentially useful as therapeutic agents for the treatmentof cancer.

[0137] These results indicate that variation of substituents on C-2 inthe parent 19-nor-1α,25-dihydroxyvitamin D₃ can change completely (andselectively) the biological potency of the analogs. These resultssuggest that 2α-methyl-19-nor-20S-1,25-(OH)₂D₃ has preferential activityon bone, making it a candidate for treatment of bone disease.

EXAMPLE 3

[0138] Preparation of 20(S)-1α,25-Dihydroxy-2α- and20(S)-1α,25-Dihydroxy-2α-methyl-26,27-dihomo-19-norvitamin D₃ (36 and37). Reference is made to SCHEME IV.

[0139] 20(S)-25-[(Triethylsilyl)oxy]-des-A,B-26,27-dihomocholestan-8-one(32). To a solution of 20(S)-25-hydroxy Grundmann's ketone analog 31(Tetrionics, Madison, Wis.; 18.5 mg, 0.06 mmol) in anhydrous CH₂Cl₂ (60μL) was added 2,6-lutidine (17.4 μL, 0.15 mmol) and triethylsilyltrifluoromethanesulfonate (20.3 μL, 0.09 mmol). The mixture was stirredat room temperature under argon for 1 h. Benzene was added and water,and the organic layer was separated, washed with sat. CuSO₄ and water,dried (MgSO₄) and evaporated. The oily residue was redissolved in hexaneand applied on a silica Sep-Pak cartridge (2 g). Elution with hexane (10mL) gave a small quantity of less polar compounds; further elution withhexane/ethyl acetate (9:1) provided the silylated ketone. Finalpurification was achieved by HPLC (10-mm×25-cm Zorbax-Sil column, 4mL/min) using hexane/ethyl acetate (95:5) solvent system. Pure protectedhydroxy ketone 32 (16.7 mg, 66%) was eluted at R_(V) 37 mL as acolorless oil: ¹H NMR (CDCl₃) δ 0.573 (6H, q, J=7.9 Hz, 3× SiCH₂), 0.639(3H, s, 18-H₃), 0.825 (6H, t, J=7.5 Hz, 26- and 27-CH₃), 0.861 (3H, d,J=6.1 Hz, 21-H₃), 0.949 (9H, t, J=7.9 Hz, 3× SiCH₂CH₃), 2.45 (1H, dd,J=11.4, 7.6 Hz, 14α-H).

[0140] 20(S)-1α,25-Dihydroxy-2-methylene-26,27-dihomo-19-norvitamin D₃(35). To a solution of phosphine oxide 33 (9.1 mg, 15.6 μmol) inanhydrous THF (150 μL) at 0° C. was slowly added n-BuLi (2.5 M inhexanes, 7 μL, 17.5 μmol) under argon with stirring. The solution turneddeep orange. It was stirred for 10 min at 0° C., then cooled to -78° C.and a precooled (−78° C.) solution of protected hydroxy ketone 32 (16.5mg, 39.0 μmol) in anhydrous THF (300+100 μL) was slowly added. Themixture was stirred under argon at −78° C. for 1.5 h and at 0° C. for 19h. Water and ethyl acetate were added, and the organic phase was washedwith brine, dried (MgSO₄) and evaporated. The residue was dissolved inhexane and applied on a silica Sep-Pak cartridge, and washed withhexane/ethyl acetate (99.7:0.3, 20 mL) to give slightly impure19-norvitamin derivative 34 (ca. 4 mg). The Sep-Pak was then washed withhexane/ethyl acetate (96:4, 10 mL) to recover some unchanged C,D-ringketone (contaminated with, 14-isomer), and with ethyl acetate (10 mL) torecover diphenylphosphine oxide 33 (ca. 6 mg) that was subsequentlypurified by HPLC (10-mm×25-cm Zorbax-Sil column, 4 mL/min) usinghexane/2-propanol (9:1) solvent system; pure compound 33 (5.1 mg) waseluted at R_(V) 36 mL. The protected vitamin 34 was further purified byHPLC (6.2-mm×25-cm Zorbax-Sil column, 4 mL/min) using hexane/ethylacetate (99.9:0.1) solvent system. Pure compound 34 (3.6 mg, 67% yieldconsidering the recovery of unreacted 33) was eluted at R_(V) 19 mL as acolorless oil: UV (in hexane) )max 244.0, 252.5, 262.5 nm; ¹H NMR(CDCl₃) δ 0.026, 0.048, 0.066, and 0.079 (each 3H, each s, 4× SiCH₃),0.544 (3H, s, 18-H₃), 0.570 (6H, q, J=7.9 Hz, 3× SiCH₂), 0.821 (6H, t,J=7.5 Hz, 26- and 27-CH₃), 0.849 (3H, d, J=6.7 Hz, 21-H₃), 0.864 and0.896 (9H and 9H, each s, 2× Si-t-Bu), 0.946 (9H, t, J=7.9 Hz, 3×SiCH₂CH₃), 1.99 (2H, m), 2.18 (1H, dd, J=12.6, 8.2 Hz, 4β-H), 2.34 (1H,dd, J=13.0, 2.9 Hz, 100-H), 2.46 (1H, dd, J=12.6,4.3 Hz, 4α-H), 2.51(1H, dd, J=13.0, 6.2 Hz, 10α-H), 2.82 (1H, br d, J=12 Hz, 9β-H), 4.43(2H, m, 1β- and 3α-H), 4.92 and 4.97 (1H and 1H, each s, ═CH₂), 5.84 and6.22 (1H and 1H, each d, J=11.2 Hz, 7- and 6-H); MS m/z (relativeintensity) 786 (M⁺, 15), 757 (M⁺-Et, 22), 729 (M⁺-t-Bu, 5), 654 (100),522 (15), 366 (43), 201 (31).

[0141] Protected vitamin 34 (3.5 mg) was dissolved in benzene (150 μL)and the resin (AG 50W-X4,40 mg; prewashed with methanol) in methanol(550 μL) was added. The mixture was stirred at room temperature underargon for 14 h, diluted with ethyl acetate/ether (1:1, 4 mL) anddecanted. The resin was washed with ether (8 mL) and the combinedorganic phases washed with brine and saturated NaHCO₃, dried (MgSO₄) andevaporated. The residue was purified by HPLC (6.2-mm×25-cm Zorbax-Silcolumn, 4 mL/min) using hexane/2-propanol (9:1) solvent system.Analytically pure 2-methylene-19-norvitamin 35 (1.22 mg, 62%) wascollected at R_(V) 21 mL as a white solid: UV (in EtOH) λ_(max) 243.5,252.0, 262.0 nm; ¹H NMR (CDCl₃) δ 0.550 (3H, s, 18-H₃), 0.855 (3H, d,J=6.8 Hz, 21-H₃), 0.860 (6H, t, J=7.5 Hz, 26- and 27-CH₃), 2.00 (3H, m),2.30 (1H, dd, J=13.3, 8.6 Hz, 10α-H), 2.33 (1H, dd, J=13.3, 6.3 Hz,41-H), 2.58 (1H, dd, J=13.3, 3.9 Hz, 4α-H), 2.82 (1H, br d, J=12 Hz,9β-H), 2.85 (1H, dd, J=13.3, 4.7 Hz, 10β-H), 4.48 (2H, m, 1β- and 3α-H),5.09 and 5.11 (1H and 1H, each s, ═CH₂), 5.89 and 6.36 (1H and 1H, eachd, J=11.3 Hz, 7- and 6-H); MS m/z (relative intensity) 444 (M⁺, 100),426 (35), 408 (11), 397 (19), 379 (32), 341 (31), 287 (32), 273 (43),269 (28), 251 (22); exact mass calcd for C₂₉H₄₈O₃ 444.3603, found444.3602.

[0142] 20(S)-1α,25-Dihydroxy-2α- and20(S)-1α,25-Dihydroxy-2β-methyl-26,27-dihomo-19-norvitamin D₃ (36 and37). Tris(triphenylphosphine)rhodium (1) chloride (2.3 mg, 2.5 μmol) wasadded to dry benzene (2.5 mL) presaturated with hydrogen. The mixturewas stirred at room temperature until a homogeneous solution was formed(ca. 45 min). A solution of vitamin 35 (1.0 mg, 2.3 μmol) in dry benzene(0.5 mL) was then added and the reaction was allowed to proceed under acontinuous stream of hydrogen for 4.5 h. A new portion of the catalyst(2.3 mg, 2.5 μmol) was added and hydrogen was passed for additional 1 h.Benzene was removed under vacuum, the residue was redissolved inhexane/ethyl acetate (1:1, 2 mL) and applied on Waters silica Sep-Pak. Amixture of 2-methyl vitamins was eluted with the same solvent system (20mL). The compounds were further purified by HPLC (6.2 mm×25 cmZorbax-Sil column, 4 mL/min) using hexane/2-propanol (9:1) solventsystem. The mixture (ca. 1:1) of 2-methyl-19-norvitamins 36 and 37 (0.37mg, 37%) gave a single peak at R_(V) 23 mL. Separation of both epimerswas achieved by reversed-phase HPLC (6.2-mm×25-cm Zorbax-ODS column, 2mL/min) using methanol/water (85:15) solvent system. 20-Methyl vitamin37 was collected at R_(V) 21 mL and its 2α-epimer 36 at R_(V) 27 mL.

[0143] 36: UV (in EtOH) λ_(max) 242.5, 251.0, 261.0; ¹H NMR (CDCl₃) δ0.534 (3H, s, 18-H₃), 0.851 (3H, d, J˜7 Hz, 21-H₃), 0.858 (6H, t, J=7.5Hz, 26- and 27-CH₃), 1.133 (3H, d, J=6.9 Hz, 2α-CH₃), 2.13 (1H, ˜t, J˜12Hz, 40-H), 2.23 (1H, br d, J=13.4 Hz, 10β-H), 2.60 (1H, dd, J=13.1, 4.4Hz, 4α-H), 2.80 (2H, m, 9,- and 100α-H), 3.61 (1H, m, w/2=26 Hz, 3α-H),3.96 (1H, m, w/2=13 Hz, 1β-H), 5.82 and 6.37 (1H and 1H, each d, J=11.2Hz, 7- and 6-H); MS m/z (relative intensity) 446 (M⁺, 53), 428 (46), 410(12), 399 (35), 381 (17), 289 (35), 273 (48), 271 (30), 253 (24), 69(100); exact mass calcd for C₂₉H₅₀O₃ 446.3760, found 446.3758.

[0144] 37: UV (in EtOH) λ_(max) 242.5, 251.0, 261.0 nm; ¹H NMR (CDCl₃) δ0.546 (3H, s, 18-H₃), 0.855 (3H, d, J=6.8 Hz, 21-H₃), 0.860 (6H, t,J=7.4 Hz, 26- and 27-CH₃), 1.143 (3H, d, J=6.9 Hz, 2β-CH₃), 2.34 (1H,dd, J=13.7, 3.3 Hz, 4,-H), 2.43 (1H, br d, J=13.7 Hz, 4α-H), 2.80 (1H,dd, J=12 and 4 Hz, 9β-H), 3.08 (1H, dd, J=12.8, 4.1 Hz, 10β-H), 3.50(1H, m, w/2=26 Hz, 1β-H), 3.90 (1H, m, w/2=11 Hz, 3α-H), 5.87 and 6.26(1H and 1H, each d, J=11.2 Hz, 7- and 6-H); MS m/z (relative intensity)446 (M⁺, 39) 428 (46), 410 (12), 399 (30), 381 (17), 289 (37), 273 (50),271 (31), 253 (27), 69 (100); exact mass calcd for C₂₉H₅₀O₃ 446.3760,found 446.3740.

Biological Activity of20(S)-1α,25-Dihydroxy-2α-methyl-26,27-dihomo-19-nor-vitamin D₃ and20(S)-1α,25-Dihydroxy-2β-methyl-26,27-dihomo-19-norvitamin D₃ (36 and37)

[0145] Competitive binding of the analogs to the porcine intestinalreceptor was carried out by the method described by Dame et alBiochemistry 25, 4523-4534, 1986).

[0146] The differentiation of HL-60 promyelocytic into monocytes wasdetermined as described by Ostrem et al (J. Biol. Chem. 262 14164-14171,1987). TABLE 6 VDR Binding Properties^(a) and HL-60 DifferentiatingActivities^(b) of 2- Substituted Analogs of20(S)-1α,25-Dihydroxy-26,27-dihomo- 19-norvitamin D₃ VDR Binding HL-60Differentiation Compd. ED₅₀ Binding ED₅₀ Activity Compound no. (M) ratio(M) ratio 1α,25-(OH)₂D₃ 8.7 × 10⁻¹⁰ 1 4.0 × 10⁻⁹ 12α-methyl-26,27-dihomo- 36 3.1 × 10⁻⁹ 3.6 6.0 × 10⁻¹¹ 0.0119-nor-20(S)-1α,25-(OH)₂D₃ 2β-methyl-26,27-dihomo- 37 4.8 × 10⁻⁹ 5.5 1.1× 10⁻¹⁰ 0.03 19-nor-20(S)-1α,25-(OH)₂D₃ # ED₅₀ to the ED₅₀ for1α,25-(OH)₂D₃. # ratio of the analog average ED₅₀ to the ED₅₀ for1α,25-(OH)₂D₃.

[0147] TABLE 7 Support of Intestinal Calcium Transport and Bone CalciumMobilization by 2-Substituted Analogs of of 20(S)-1α,25-Dihydroxy-26,27-dihomo-19-norvitamin D₃ in Vitamin D-Deficient Ratson a Low-Calcium Diet^(a) Ca Transport Serum Ca Compd. Amount S/M (mean± (mean ± Compound no. (pmol) SEM) SEM) one (control) 0 2.7 ± 0.3^(b)4.7 ± 0.2^(b) 1α,25-(OH)₂D₃ 260 7.2 ± 0.6^(c) 5.6 ± 0.2^(c)2α-methyl-26, 36 32 5.8 ± 0.4^(d) ¹ 5.9 ± 0.2^(d) ¹ 27-dihomo-19-nor-20(S)-1α, 65 8.4 ± 0.8^(d) ² 9.3 ± 0.2^(d) ² 25-(OH)₂D₃ none(control) 0 3.6 ± 0.4^(b) 5.0 ± 0.1^(b) 1α,25-(OH)₂D₃ 260 5.0 ± 0.4^(c)6.3 ± 0.2^(c) 2β-methyl-26, 37 65 4.7 ± 0.6^(d) ¹ 5.0 ± 0.0^(d) ¹27-dihomo- 19-nor-20(S)-1α, 260 5.2 ± 0.6^(d) ² 9.9 ± 0.3^(d) ²25-(OH)₂D₃ # analysis was done by Student's t-test. Statistical data:serosal/mucosal (S/M), panel 1, b from c, and d², p < 0.001, b from d¹,p = 0.001; panel 2, b from c, d¹, and d²,p < 0.05; serum calcium, panel1, b from c and d¹, p < 0.05, b from d², p < 0.001; panel 2, b from c, p< 0.01, b from d¹, NS, b from d², p < 0.001.

EXAMPLE 4

[0148] Preparation of20(S)-1α,25-Dihydroxy-26,27-dimethylene-2α-methyl-19-norvitamin D₃ and20(S)-1α,25-Dihydroxy-26,27-dimethylene-2β-methyl-19-norvitamin D₃ (48and 49). Reference is made to SCHEMES V and VI.

[0149]20(S)-25-[(Triethylsilyl)oxy]-des-A,B-26,27-dimethylene-cholestan-8-one(42). To a solution of 20(8)-25-hydroxy Grundmann's ketone analog 41(Tetrionics, Madison, WI; 15.0 mg, 0.049 mmol) in anhydrous CH₂Cl₂ (50μL) was added 2,6-lutidine (15 μL, 0.129 mmol) and triethylsilyltrifluoromethanesulfonate (17.0 μL, 0.075 mmol). The mixture was stirredat room temperature under argon for 1 h. Benzene was added and water,and the organic layer was separated, washed with sat. CuSO₄ and water,dried (MgSO₄) and evaporated. The oily residue was redissolved in hexaneand applied on a silica Sep-Pak cartridge (2 g). Elution with hexane (10mL) gave a small quantity of less polar compounds; further elution withhexane/ethyl acetate (9:1) provided the silylated ketone. Finalpurification was achieved by HPLC (10-mm×25-cm Zorbax-Sil column, 4mL/min) using hexane/ethyl acetate (95:5) solvent system. Pure protectedhydroxy ketone 42 (9.4 mg, 46%) was eluted at R_(V) 39 mL as a colorlessoil: ¹H NMR (CDCl₃) δ 0.576 (6H, q, J=7.9 Hz, 3× SiCH₂), 0.638 (3H, s,18-H₃), 0.865 (3H, d, J=6.1 Hz, 21-H₃), 0.949 (9H, t, J=7.9 Hz, 3×SiCH₂CH₃), 2.45 (1H, dd, J=11.4, 7.5 Hz, 14α-H).

[0150] 20(S)-1α,25-Dihydroxy-26,27-dimethylene-2-methylene-19-norvitaminD₃ (47). To a solution of phosphine oxide 43 (17.7 mg, 30.4 μmol) inanhydrous THF (300 μL) at 0° C. was slowly added n-BuLi (2.5 M inhexanes, 13 μL, 32.5 μmol) under argon with stirring. The solutionturned deep orange. It was stirred for 10 min at 0° C., then cooled to−78° C. and a precooled (−78° C.) solution of protected hydroxy ketone41 (17.8 mg, 42.3 μmol) in anhydrous THF (300+100 μL) was slowly added.The mixture was stirred under argon at −78° C. for 1.5 h and at 0° C.for 18 h. Water and ethyl acetate were added, and the organic phase waswashed with brine, dried (MgSO₄) and evaporated. The residue wasdissolved in hexane and applied on a silica Sep-Pak cartridge, andwashed with hexanelethyl acetate (99.7:0.3, 20 mL) to give slightlyimpure 19-norvitamin derivative 44 (ca. 11 mg). The Sep-Pak was thenwashed with hexane/ethyl acetate (96:4, 10 mL) to recover some unchangedC,D-ring ketone (contaminated with 14β-isomer), and with ethyl acetate(10 mL) to recover diphenylphosphine oxide 43 (ca. 8 mg) that wassubsequently purified by HPLC (10-mm×25-cm Zorbax-Sil column, 4 mL/min)using hexane/2-propanol (9:1) solvent system; pure compound 43 (7.6 mg)was eluted at R_(V) 36 mL. The protected vitamin 44 was further purifiedby HPLC (6.2-mm×25-cm Zorbax-Sil column, 4 mL/min) using hexane/ethylacetate (99.9:0.1) solvent system. Pure compound 44 (10.1 mg, 74% yieldconsidering the recovery of unreacted 43) was eluted at R_(V) 27 mL as acolorless oil: UV (in hexane) λ_(max) 244.0, 252.5, 262.5 nm; ¹H NMR(CDCl₃) δ 0.027, 0.048, 0.067, and 0.080 (each 3H, each s, 4× SiCH₃),0.544 (3H, s, 18-H₃), 0.575 (6H, q, J=7.9 Hz, 3× SiCH₂), 0.854 (3H, d,J=6.1 Hz, 21-H₃), 0.866 and 0.896 (9H and 9β-H, each s, 2× Si-t-Bu),0.947 (9H, t, J=7.9 Hz, 3× SiCH₂CH₃), 1.99 (2H, m), 2.18 (1H, dd,J=12.8, 8.6 Hz, 40-H), 2.34 (1H, dd, J=13.2, 2.7 Hz, 10w-H), 2.46 (1H,dd, J=12.8, 4.4 Hz, 4α-H), 2.51 (11H, dd, J=13.2, 6.0 Hz, 10α-H), 2.82(1H, br d, J=12 Hz, 9β-H), 4.42 (2H, m, 1β- and 3α-H), 4.92 and 4.97 (1Hand 1H, each s, ═CH₂), 5.84 and 6.22 (1H and 1H, each d, J=11.2 Hz, 7-and 6-H); MS m/z (relative intensity) 784 (M⁺, 8), 755 (M⁺-Et, 4), 727(M⁺-t-Bu, 6), 652 (100), 520 (31), 366 (49), 199 (23).

[0151] Protected vitamin 44 (7.0 mg) was dissolved in benzene (220 μL)and the resin (AG 50W-X4, 95 mg; prewashed with methanol) in methanol(1.2 mL) was added. The mixture was stirred at room temperature underargon for 21 h, diluted with ethyl acetate/ether (1:1, 4 mL) anddecanted. The resin was washed with ether (10 mL) and the combinedorganic phases washed with brine and saturated NaHCO₃, dried (MgSO₄) andevaporated. The residue was separated by HPLC (6.2-mm×25-cm Zorbax-Silcolumn, 4 mL/min) using hexane/2-propanol (9:1) solvent system and thefollowing analytically pure 2-methylene-19-norvitamins were isolated:1×-hydroxy-25-dehydrovitamin 45 (0.68 mg, 17%) was collected at R_(V) 13mL, 1α-hydroxy-25-methoxyvitamin 46 (0.76 mg, 19%) was collected atR_(V) 16 mL and 1α,25-dihydroxyvitamin 47 (2.0 mg, 51%) was collected atR_(V) 21 mL.

[0152] 45: WV (in EtOH) λ_(max) 243.5, 251.5, 262.0 nm; ¹H NMR (CDCl₃) δ0.542 (3H, s, 18-H₃), 0.847 (3H, d, J 6.5 Hz, 21-H₃), 1.93-2.07 (4H, m),2.18-2.25 (2H, m), 2.26-2.36 (4H, m), 2.58 (1H, dd, J=13.3, 3.9 Hz,4α-H), 2.82 (1H, br d, J=13 Hz, 9β-H), 2.85 (1H, dd, J=13.3, 4.5 Hz,10β-H), 4.48 (2H, m, 1β- and 3α-H), 5.09 and 5.11 (1H and 1H, each s,═CH₂), 5.32 (1H, m, w/2=7 Hz, 24-H), 5.88 and 6.36 (1H and 1H, each d,J=11.1 Hz, 7- and 6-H); MS m/z (relative intensity) 424 (M⁺, 100), 406(7), 339 (16), 287 (16), 271 (24), 269 (17), 251 (12); exact mass calcdfor C₂₉H₄₄O₂ 424.3341, found 424.3343. 46: UV (in EtOH) λ_(max) 243.5,252.0, 262.0 nm; ¹H NMR (CDCl₃) δ 0.553 (3H, s, 18-H₃), 0.858 (3H, d,J=6.5 Hz, 21-H₃), 1.95-2.05 (2H, m), 2.30 (1H, dd, J=13.3, 8.3 Hz,10α-H), 2.33 (1H, dd, 3=13.4, 6.0 Hz, 4β-H), 2.58 (1H, dd, J=13.4, 3.8Hz, 4α-H), 2.82 (1H, br d, J=13 Hz, 9β-H), 2.85 (1H, dd, J=13.3, 4.4 Hz,10β-H), 3.13 (3H, s, OCH₃), 4.48 (2H, m, 1β- and 3α-H), 5.09 and 5.11(1H and 1H, each s, ═CH₂), 5.89 and 6.36 (1H and 1H, each d, J=11.2 Hz,7- and 6-H); MS m/z (relative intensity) 456 (M⁺, 54), 424 (27), 406(12), 339 (16), 287 (13), 271 (41), 99 (100); exact mass calcd forC₃₀H₄₈O₃ 456.3603, found 456.3603.

[0153] 47: UV (in EtOH) λ_(max) 243.5, 252.0, 262.0 nm; ¹H NMR (CDCl₃) δ0.551 (3H, s, 18-H₃), 0.859 (3H, d, J=6.6 Hz, 21-H₃), 1.95-2.05 (2H, m),2.30 (1H, dd, J=13.5, 8.4 Hz, 10α-H), 2.33 (1H, dd, J=13.3, 6.3 Hz,4β-H), 2.58 (1H, dd, J=13.3, 4.0 Hz, 4α-H), 2.82 (1H, br d, J=12 Hz,9β-H), 2.85 (1H, dd, J=13.5, 4.4 Hz, 10β-H), 4.48 (2H, m, 1β- and 3α-H),5.09 and 5.11 (1H and 1H, each s, ═CH₂), 5.89 and 6.36 (1H and 1H, eachd, J=11.3 Hz, 7- and 6-H); MS m/z (relative intensity) 442 (M⁺, 100),424 (47), 406 (15), 339 (34), 287 (27), 271 (42), 269 (36), 251 (26);exact mass calcd for C₂₉H₄₆O₃ 442.3447, found 442.3442.

[0154] 20(S)-1α,25-Dihydroxy-26,27-dimethylene-2α- and20(S)-1α,25-Dihydroxy-26,27-dimethylene-21β-methyl-19-norvitamin D₃ (48and 49). Tris(triphenylphosphine)rhodium (I) chloride (2.3 mg, 2.5 μmol)was added to dry benzene (2.5 mL) presaturated with hydrogen. Themixture was stirred at room temperature until a homogeneous solution wasformed (ca. 45 min). A solution of vitamin 47 (1.0 mg, 2.3 μmol) in drybenzene (0.5 mL) was then added and the reaction was allowed to proceedunder a continuous stream of hydrogen for 3 h. A new portion of thecatalyst (2.3 mg, 2.5 μmol) was added and hydrogen was passed foradditional 2 h. Benzene was removed under vacuum, the residue wasredissolved in hexane/ethyl acetate (1:1, 2 mL) and applied on Waterssilica Sep-Pak. A mixture of 2-methyl vitamins was eluted with the samesolvent system (20 mL). The compounds were further purified by HPLC (6.2mm×25 cm Zorbax-Sil column, 4 mL/min) using hexane/2-propanol (9:1)solvent system. The mixture (ca. 1:1) of 2-methyl-19-norvitamins 48 and49 (0.23 mg, 23%) gave a single peak at R_(V) 23 mL. Separation of bothepimers was achieved by reversed-phase HPLC (6.2-mm×25-cm Zorbax-ODScolumn, 2 mL/min) using methanol/water (85:15) solvent system. 2β-Methylvitamin 49 was collected at R_(V) 19 mL and its 2α-epimer 48 at R_(V) 24mL.

[0155] 48: UV (in EtOH),max 242.5, 251.0, 261.5 nm; ¹H NMR (CDCl₃) δ0.534 (3H, s, 18-H₃), 0.853 (3H, d, J=6.6 Hz, 21-H₃), 1.134 (3H, d,J=6.8 Hz, 2α-CH₃), 2.13 (1H, ˜t, J˜12 Hz, 4β-H),2.22(1H, br d, J=13 Hz,10β-H), 2.60(1H, dd, J=12.8, 4.6 Hz, 4α-H), 2.80 (2H, m, 9β- and 10α-H),3.61 (1H, m, w/2=23 Hz, 3α-H), 3.96 (1H, m, w/2=11 Hz, 1β-H), 5.82 and6.37 (1H and 1H, each d, J=11.3 Hz, 7- and 6-H); MS m/z (relativeintensity) 444 (M⁺, 84), 426 (53), 289 (36), 271 (58), 253 (19); exactmass calcd for C₂₉H₄₈O₃ 444.3603, found 444.3602.

[0156] 49: UV (in EtOH) λ_(max) 242.5, 251.0, 261.5 nm; ¹H NMR (CDCl₃) δ0.547 (3H, s, 18-H₃), 0.859 (3H, d, J=6.8 Hz, 21-H₃), 1.143 (3H, d,J=6.8 Hz, 2β-CH₃), 2.34 (1H, dd, J=13.7,-3.3 Hz, 4β-H), 2.43 (1H, br d,J=13.7 Hz, 4α-H), 2.80 (1H, br d, J=12 Hz, 9β-H), 3.08 (1H, dd, J=12.9,4.4 Hz, 10β-H), 3.50 (1H, m, w/2=25 Hz, 1β-H), 3.90 (1H, m, w/2=12 Hz,3α-H), 5.87 and 6.26 (1H and 1H, each d, J=11.3 Hz, 7- and 6-H); MS m/z(relative intensity) 444 (M⁺, 75), 426 (59), 289 (34), 271 (59), 253(18); exact mass calcd for C₂₉H₁₈O₃ 444.3603, found 444.3611.

Biological Activity of20(S)-1α,25-Dihydroxy-26,27-dimethylene-2α-methyl-19-norvitamin D₃ and20(S)-1α,25-Dihydroxy-26,27-dimethylene-2β-methyl-19-norvitamin D₃ (48and 49)

[0157] Competitive binding of the analogs to the porcine intestinalreceptor was carried out by the method described by Dame et al(Biochemistry 2, 4523-4534, 1986).

[0158] The differentiation of HL-60 promyelocytic into monocytes wasdetermined as described by Ostrem et al (J. Biol. Chem. 262,14164-14171, 1987). TABLE 8 VDR Binding Properties^(a) and HL-60Differentiating Activities^(b) of 2-Substituted Analogs of20(S)-1α,25-Dihydroxy-26,27- dimethylene-19-norvitamin D₃ VDR BindingHL-60 Differentiation Compd. ED₅₀ Binding ED₅₀ Activity Compound no. (M)ratio (M) ratio 1α,25-(OH)₂D₃ 8.7 × 10⁻¹⁰ 1 4.0 × 10⁻⁹  1 2α-methyl- 483.5 × 10⁻⁹  4.0 4.4 × 10⁻¹¹ 0.01 26,27-dim- ethylene-19- nor-20(S)-1α,25-(OH)₂D₃ 2β-methyl- 49 2.3 × 10⁻⁹  2.6 3.2 × 10⁻¹⁰ 0.08 26,27-dim-ethylene-19- nor-20(S)-1α, 25-(OH)₂D₃ # ED₅₀ for 1α,25-(OH)₂D₃. # ED₅₀to the ED₅₀ for 1α,25-(OH)₂D₃.

[0159] TABLE 9 Support of Intestinal Calcium Transport and Bone CalciumMobilization by 2-Substituted Analogs of of20(1S)-1α,25-Dihydroxy-26,27- dimethylene- 19-norvitamin D₃ in VitaminD-Deficient Rats on a Low-Calcium Diet^(a) Compd. Amount Ca TransportS/M Serum Ca Compound no. (pmol) (mean ± SEM) (mean ± SEM) none(control)  0 2.7 ± 0.3^(b) 4.7 ± 0.2^(b) 1α,25-(OH)₂D₃ 260 7.2 ± 0.6^(c)5.6 ± 0.2^(c) 2α-methyl-26,27- 48  32 7.9 ± 1.0^(d1) 6.9 ± 0.5^(d1)dimethylene- 19-nor-20(S)-1α,  65 9.0 ± 1.0^(d2) 9.0 ± 0.3^(d2)25-(OH)₂D₃ none (control)  0 3.6 ± 0.4^(b) 5.0 ± 0.1^(b) 1α,25-(OH)₂D₃260 5.0 ± 0.4^(c) 6.3 ± 0.2^(c) 2β-methyl-26,27- 49  65 4.9 ± 0.6^(d1)5.8 ± 0.2^(d1) dimethylene- 19-nor-20(S)-1α, 260 5.4 ± 0.7^(d2) 9.5 ±0.1^(d2) 25-(OH)₂D₃ # by Student's t-test. Statistical data:serosal/mucosal (S/M), panel 1 b from c, d¹, and d², p < 0.001; panel 2,b from c, d¹, and d², p < 0.05; serum calcium, panel 1, b from c, d¹, p< 0.05, b from d², p < 0.001; panel 2, b from c and d¹, p < 0.01, b fromd², p < 0.001.

[0160] For treatment purposes, the novel compounds of this inventiondefined be formula I may be formulated for pharmaceutical applicationsas a solution in innocuous solvents, or as an emulsion, suspension ordispersion in suitable solvents or carriers, or as pills, tablets orcapsules, together with solid carriers, according to conventionalmethods known in the art. Anby such formulations may also contain otherpahrmaceutically-acceptable acceptable and non-toxic excipients such asstabilizers, anti-oxidants, binders, coloring agents or emulsifying ortaste-modifying agents.

[0161] The compounds may be administered orally, topically,parenterally, sublingually, intranasally, or transdermally. Thecompounds are advantageously administered by injection or by intravenousinfusion or suitable sterile solutions, or in the form of liquid orsolid doses via the alimentary canal, or in the form of creams,ointments, patches, or similar vehicles suitable for transdermalapplications. Doses of from 0.1 g to 50 kg per day of the compounds areappropriate for treatment purposes, such doses being adjusted accordingto the disease to be treated, its severity and the response of thesubject as is well understood in the art. Since the new compoundsexhibit specificity of action, each may be suitably administered alone,or together with graded doses of another active vitamin D compound—

[0162] e.g. 1α-hydroxyvitamin D₂ or D₃, or 1α,25-dihydroxyvitamin D₃—insituations where different degrees of bone mineral mobilization andcalcium transport stimulation is found to be advantageous.

[0163] Compositions for use in the above-mentioned treatment ofpsoriasis and other malignancies comprise an effective amount of one ormore 2-substituted-19-nor-vitamin D compound as defined by the aboveformula I as the active ingredient, and a suitable carrier. An effectiveamount of such compounds for use in accordance with this invention isfrom about 0.01 μg to about 100 μg per gm of composition, and may beadministered topically, transdermally, orally, sublingually,intranasally, or parenterally in dosages of from about 0.1 μg/day toabout 100 μg/day.

[0164] The compounds may be formulated as creams, lotions, ointments,topical patches, pills, capsules or tablets, or in liquid form assolutions, emulsions, dispersions, or suspensions in pharmaceuticallyinnocuous and acceptable solvent or oils, and such preparations maycontain in addition other pharmaceutically innocuous or beneficialcomponents, such as stabilizers, antioxidants, emulsifiers, coloringagents, binders or taste-modifying agents.

[0165] The compounds are advantageously administered in amountssufficient to effect the differentiation of promyelocytes to normalmacrophages. Dosages as described above are suitable, it beingunderstood that the amounts given are to be adjusted in accordance withthe severity of the disease, and the condition and response of thesubject as is well understood in the art.

[0166] The formulations of the present invention comprise an activeingredient in association with a pharmaceutically acceptable carriertherefore and optionally other therapeutic ingredients. The carrier mustbe “acceptable” in the sense of being compatible with the otheringredients of the formulations and not deleterious to the recipientthereof.

[0167] Formulations of the present invention suitable for oraladministration may be in the form of discrete units as capsules,sachets, tablets or lozenges, each containing a predetermined amount ofthe active ingredient; in the form of a powder or granules; in the formof a solution or a suspension in an aqueous liquid or non-aqueousliquid; or in the form of an oil-in-water emulsion or a water-in-oilemulsion.

[0168] Formulations for rectal administration may be in the form of asuppository incorporating the active ingredient and carrier such ascocoa butter, or in the form of an enema.

[0169] Formulations suitable for parenteral administration convenientlycomprise a sterile oily or aqueous preparation of the active ingredientwhich is preferably isotonic with the blood of the recipient.

[0170] Formulations suitable for topical administration include liquidor semi-liquid preparations such as liniments, lotions, applicants,oil-in-water or water-in-oil emulsions such as creams, ointments orpastes; or solutions or suspensions such as drops; or as sprays.

[0171] For asthma treatment, inhalation of powder, self-propelling orspray formulations, dispensed with a spray can, a nebulizer or anatomizer can be used. The formulations, when dispensed, preferably havea particle size in the range of 10 to 100μ.

[0172] The formulations may conveniently be presented in dosage unitform and may be prepared by any of the methods well known in the art ofpharmacy. By the term “dosage unit” is meant a unitary, i.e. a singledose which is capable of being administered to a patient as a physicallyand chemically stable unit dose comprising either the active ingredientas such or a mixture of it with solid or liquid pharmaceutical diluentsor carriers.

[0173] In its broadest application, the present invention relates to any19-nor-2-alkyl analogs of vitamin D which have the vitamin D nucleus. Byvitamin D nucleus, it is meant a central part consisting of asubstituted chain of five carbon atoms which correspond to positions 8,14, 13, 17 and 20 of vitamin D, and at the ends of which are connectedat position 20 a structural moiety representing any of the typical sidechains known for vitamin D type compounds (such as R as previouslydefined herein), and at position 8 the 5,7-diene moiety connected to theA-ring of an active 1α-hydroxy vitamin D analog (as illustrated byformula I herein). Thus, various known modifications to the six-memberedC-ring and the five-membered D-ring typically present in vitamin D, suchas the lack of one or the other or both, are also embraced by thepresent invention.

[0174] Accordingly, compounds of the following formulae Ia, are alongwith those of formula I, also encompassed by the present invention:

[0175] In the above formula Ia, the definitions of Y₁, Y₂, R₆, and Z areas previously set forth herein. With respect to X₁, X₂, X₃, X₄, X₅, X₆,X₇, X₈ and X₉, these substituents may be the same or different and areselected from hydrogen or lower alkyl, i.e. a C₁₋₅ alkyl such as methyl,ethyl or n-propyl. In addition, paired substituents X₁ and X₄ or X₅, X₂or X₃ and X₆ or X₇; X₄ or X₅ and X₈ or X₉, when taken together with thethree adjacent carbon atoms of the central part of the compound, whichcorrespond to positions 8, 14, 13 or 14,, 13, 17 or 13, 17, 20respectively, can be the same or different and form a saturated orunsaturated, substituted or unsubstituted, carbocyclic 3, 4, 5, 6 or 7membered ring.

[0176] Preferred compounds of the present invention may be representedby one of the following formulae:

[0177] In the above formulae Ib, Ic, Id, Ie, If, Ig and Ih, thedefinitions of Y₁, Y₂, R₆, R, Z, X₁, X₂, X₃, X₄, X₅, X₆, X₇ and X₈ fareas previously set forth herein. The substituent Q represents a saturatedor unsaturated, substituted or unsubstituted, hydrocarbon chaincomprised of 0, 1, 2, 3 or 4 carbon atoms, but is preferably the group—(CH₂)_(k)— where k is an integer equal to 2 or 3.

[0178] Methods for making compounds of formulae Ia-Ih are known.Specifically, reference is made to International Application NumberPCT/FP94/02294 filed Jul. 7, 1994 and published Jan. 19, 1995 underInternational Publication Number WO95/01960.

I claim:
 1. A compound having the formula:

where Y₁ and Y₂, which may be the same or different, are each selectedfrom the group consisting of hydrogen and a hydroxy-protecting group, R₆is selected from alkyl, hydroxyalkyl and fluoroalkyl, and where thegroup R is represented by the structure:

where the stereochemical center at carbon 20 may have the R or Sconfiguration, and where Z is selected from Y, —OY, —CH₂OY, —C≡CY,—CH═CHY, and —CH₂CH₂CH═CR³R⁴, where the double bond may have the cis ortrans geometry, and where Y is selected from hydrogen, methyl, —COR⁵ anda radical of the structure:

where m and n, independently, represent the integers from 0 to 5, whereR¹ is selected from hydrogen, deuterium, hydroxy, protected hydroxy,fluoro, trifluoromethyl, and C₁₋₅-alkyl, which may be straight chain orbranched and, optionally, bear a hydroxy or protected-hydroxysubstituent, and where each of R², R³, and R⁴, independently, isselected from deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyland C₁₋₅ alkyl, which may be straight-chain or branched, and optionally,bear a hydroxy or protected-hydroxy substituent, and where R¹ and R²,taken together, represent an oxo group, or an alkylidene group, ═CR²R³,or the group —(CH₂)_(p)—, where p is an integer from 2 to 5, and whereR³ and R⁴, taken together, represent an oxo group, or the group—(CH₂)_(q)—, where q is an integer from 2 to 5, and where R⁵ representshydrogen, hydroxy, protected hydroxy, C₁₋₅ alkyl or —OR⁷ where R⁷represents C₁₋₅ alkyl, and wherein any of the CH-groups at positions 20,22, or 23 in the side chain may be replaced by a nitrogen atom, or whereany of the groups —CH(CH₃)—, —CH(R³)—, or —CH(R²)— at positions 20, 22,and 23, respectively, may be replaced by an oxygen or sulfur atom. 2.The compound of claim 1 where R is a side chain of the formula


3. The compound of claim 1 where R is a side chain of the formula


4. 19-nor-26,27-dihomo-20(S)-2α-methyl-1α,25-dihydroxyvitamin D₃. 5.19-nor-26,27-dihomo-20(S)-2β-methyl-1α,25-dihydroxyvitamin D₃. 6.19-nor-26,27-dimethylene-20(S)-2α-methyl-1α,25-dihydroxyvitamin D₃. 7.19-nor-26,27-dimethylene-20(S)-2β-methyl-1α,25-dihydroxyvitamin D₃.
 8. Apharmaceutical composition containing at least one compound as claimedin claim 1 together with a pharmaceutically acceptable excipient.
 9. Thepharmaceutical composition of claim 8 containing19-nor-26,27-dihomo-20(S)-2α-methyl-1α,25-dihydroxyvitamin D₃ in anamount from about 0.1 μg to about 50 μg.
 10. The pharmaceuticalcomposition of claim 8 containing19-nor-26,27-dihomo-20(S)-2β-methyl-1α,25-dihydroxyvitamin D₃ in anamount from about 0.1 μg to about 50 μg.
 11. The pharmaceuticalcomposition of claim 8 containing19-nor-26,27-dimethylene-20(S)-2α-methyl-1α,25-dihydroxyvitamin D₃ in anamount from about 0.1 μg to about 50 μg.
 12. The pharmaceuticalcomposition of claim 8 containing19-nor-26,27-dimethylene-20(S)-2β-methyl-1α,25-dihydroxyvitamin D₃ in anamount from about 0.1 μg to about 50 μg.
 13. A method of treatingmetabolic bone disease where it is desired to maintain or increase bonemass comprising administering to a patient with said disease aneffective amount of a compound having the formula:

where Y₁ and Y₂, which may be the same or different, are each selectedfrom the group consisting of hydrogen and a hydroxy-protecting group, R₆is selected from all, hydroxyalkyl and fluoroalkyl, and where the groupR is represented by the structure:

where the stereochemical center at carbon 20 may have the R or Sconfiguration, and where Z is selected from Y, —OY, —CH₂OY, —C≡CY,—CH═CHY, and —CH₂CH₂CH═CR³R⁴, where the double bond may have the cis ortrans geometry, and where Y is selected from hydrogen, methyl, —COR⁵ anda radical of the structure:

where m and n, independently, represent the integers from 0 to 5, whereR¹ is selected from hydrogen, deuterium, hydroxy, protected hydroxy,fluoro, trifluoromethyl, and C₁₋₅-alkyl, which may be straight chain orbranched and, optionally, bear a hydroxy or protected-hydroxysubstituent, and where each of R², R³, and R⁴, independently, isselected from deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyland C₁₋₅ alkyl, which may be straight-chain or branched, and optionally,bear a hydroxy or protected-hydroxy substituent, and where R¹ and R²,taken together, represent an oxo group, or an alkylidene group, ═CR²R³,or the group —(CH₂)_(p)—, where p is an integer from 2 to 5, and whereR³ and R⁴, taken together, represent an oxo group, or the group—(CH₂)_(q)—, where q is an integer from 2 to 5, and where R⁵ representshydrogen, hydroxy, protected hydroxy, C₁₋₅ alkyl or —OR⁷ where R⁷represents C₁₋₅ alkyl, and wherein any of the CH-groups at positions 20,22, or 23 in the side chain may be replaced by a nitrogen atom, or whereany of the groups —CH(CH₃)—, —CH(R³)—, or —CH(²)— at positions 20, 22,and 23, respectively, may be replaced by an oxygen or sulfur atom. 14.The method of claim 13 where the disease is senile osteoporosis.
 15. Themethod of claim 13 where the disease is postmenopausal osteoporosis. 16.The method of claim 13 where the disease is steroid-inducedosteoporosis.
 17. The method of claim 13 where the disease is low boneturnover osteoporosis.
 18. The method of claim 13 where the disease isosteomalacia.
 19. The method of claim 13 where the disease is renalosteodystrophy.
 20. The method of claim 13 wherein the compound isadministered orally.
 21. The method of claim 13 wherein the compound isadministered parenterally.
 22. The method of claim 13 wherein thecompound is administered transdermally.
 23. The method of claim 13wherein the compound is administered in a dosage of from 0.1 μg to 50 μgper day.
 24. The method of claim 13 wherein the compound is19-nor-26,27-dihomo-20(S)-2α-methyl-1α,25-dihydroxyvitamin D₃.
 25. Themethod of claim 13 wherein the compound is19-nor-26,27-dihomo-20(S)-2β-methyl-1α,25-dihydroxyvitamin D₃.
 26. Themethod of claim 13 wherein the compound is19-nor-26,27-dimethylene-20(S)-2α-methyl-1α,25-dihydroxyvitamin D₃. 27.The method of claim 13 wherein the compound is19-nor-26,27-dimethylene-20(S)-2β-methyl-1α,25-dihydroxyvitamin D₃. 28.A method of treating psoriasis comprising administering to a patientwith said disease an effective amount of a compound having the formula:

where Y₁ and Y₂, which may be the same or different, are each selectedfrom the group consisting of hydrogen and a hydroxy-protecting group, R⁶is selected from alkyl, hydroxyalkyl and fluoroalkyl, and where thegroup R is represented by the structure:

where the stereochemical center at carbon 20 may have the R or Sconfiguration, and where Z is selected from Y, —OY, —CH₂OY, —C≡CY,—CH═CHY, and —CH₂CH₂CH═CR³R⁴, where the double bond may have the cis ortrans geometry, and where Y is selected from hydrogen, methyl, —COR⁵ anda radical of the structure:

where m and n, independently, represent the integers from 0 to 5, whereR¹ is selected from hydrogen, deuterium, hydroxy, protected hydroxy,fluoro, trifluoromethyl, and C₁₋₅-alkyl, which may be straight chain orbranched and, optionally, bear a hydroxy or protected-hydroxysubstituent, and where each of R², R³, and R⁴, independently, isselected from deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyland C₁₅ alkyl, which may be straight-chain or branched, and optionally,bear a hydroxy or protected-hydroxy substituent, and where R¹ and R²,taken together, represent an oxo group, or an alkylidene group, ═CR²R³,or the group —(CH₂)_(p)—, where p is an integer from 2 to 5, and whereR³ and R⁴, taken together, represent an oxo group, or the group—(CH₂)_(q)—, where q is an integer from 2 to 5, and where R⁵ representshydrogen, hydroxy, protected hydroxy, C₁₋₅ alkyl or —OR⁷ where R⁷represents C₁₌₅ alkyl, and wherein any of the CH-groups at positions 20,22, or 23 in the side chain may be replaced by a nitrogen atom, or whereany of the groups —CH(CH₃)—, —CH(R³)—, or —CH(R²)— at positions 20, 22,and 23, respectively, may be replaced by an oxygen or sulfur atom. 29.The method of claim 28 wherein the compound is19-nor-26,27-dihomo-20(S)-2α-methyl-1α,25-dihydroxyvitamin D₃.
 30. Themethod of claim 28 wherein the compound is19-nor-26,27-dihomo-20(S)-2β-methyl-1α,25-dihydroxyvitamin D₃.
 31. Themethod of claim 28 wherein the compound is19-nor-26,27-dimethylene-20(S)-2α-methyl-1α,25-dihydroxyvitamin D₃. 32.The method of claim 28 wherein the compound is19-nor-26,27-dimethylene-20(S)-2β-methyl-1α,25-dihydroxyvitamin D₃. 33.The method of claim 28 wherein said effective amount comprises about0.01 μg/day to about 100 μg/day of said compound.
 34. A method oftreating a cancerous disease comprising administering to a patient withsaid disease an effective amount of a compound having the formula:

where Y₁ and Y₂, which may be the same or different, are each selectedfrom the group consisting of hydrogen and a hydroxy-protecting group, R⁶is selected from alkyl, hydroxyalkyl and fluoroalkyl, and where thegroup R is represented by the structure:

where the stereochemical center at carbon 20 may have the R or Sconfiguration, and where Z is selected from Y, —OY, —CH₂OY, —C≡CY,—CH═CHY, and —CH₂CH₂CH═CR³R⁴, where the double bond may have the cis ortrans geometry, and where Y is selected from hydrogen, methyl, —COR⁵ anda radical of the structure:

where m and n, independently, represent the integers from 0 to 5, whereR₁ is selected from hydrogen, deuterium, hydroxy, protected hydroxy,fluoro, trifluoromethyl, and C₁₋₅-alkyl, which may be straight chain orbranched and, optionally, bear a hydroxy or protected-hydroxysubstituent, and where each of R², R³, and R⁴, independently, isselected from deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyland C₁₋₅ alkyl, which may be straight-chain or branched, and optionally,bear a hydroxy or protected-hydroxy substituent, and where R¹ and R²,taken together, represent an oxo group, or an alkylidene group, ═CR²R³,or the group —(CH₂)_(p)—, where p is an integer from 2 to 5, and whereR³ and R⁴, taken together, represent an oxo group, or the group—(CH₂)_(q)— where q is an integer from 2 to 5, and where R⁵ representshydrogen, hydroxy, protected hydroxy, C₁₋₅ alkyl or —OR⁷ where R⁷represents C₁₋₅ alkyl, and wherein any of the CH-groups at positions 20,22, or 23 in the side chain may be replaced by a nitrogen atom, or whereany of the groups —CH(CH₃)—, —CH(R³)—, or —CH(R²)— at positions 20, 22,and 23, respectively, may be replaced by an oxygen or sulfur atom. 35.The method of claim 34 where the disease is leukemia.
 36. The method ofclaim 34 where the disease is colon cancer.
 37. The method of claim 34where the disease is breast cancer.
 38. The method of claim 34 where thedisease is prostate cancer.
 39. The method of claim 34 wherein thecompound is administered orally.
 40. The method of claim 34 wherein thecompound is administered parenterally.
 41. The method of claim 34wherein the compound is administered transdermally.
 42. The method ofclaim 34 wherein the compound is administered in a dosage of from 0.1 μgto 50 μg per day.
 43. The method of claim 34 wherein the compound is19-nor-26,27-dihomo-20(S)-2α-methyl-1α,25-dihydroxyvitamin D₃.
 44. Themethod of claim 34 wherein the compound is19-nor-26,27-dihomo-20(S)-2β-methyl-1α,25-dihydroxyvitamin D₃.
 45. Themethod of claim 34 wherein the compound is19-nor-26,27-dimethylene-20(S)-2α-methyl-1α,25-dihydroxyvitamin D₃. 46.The method of claim 34 wherein tie compound is19-nor-26,27-dimethylene-20(S)-2β-methyl-1α,25-dihydroxyvitamin D₃.